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991.
An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (Tm = 58 °C), enthalpy (ΔHm = 50 ± 5 kcal mol− 1), entropy (ΔSm = 150 ± 10 cal mol− 1 K− 1), and average cooperative melting unit (〈nc〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (Tm = 40 °C; ΔHm = 27 ± 2 kcal mol− 1; ΔSm = 86 ± 6 cal mol− 1 K− 1) and noncooperative unfolding (〈nc〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell. 相似文献
992.
Alteration of the Ileal Microbiota of Weanling Piglets by the Growth-Promoting Antibiotic Chlortetracycline 总被引:1,自引:0,他引:1
Elizabeth Rettedal Sébastien Vilain Stacy Lindblom Kelly Lehnert Clay Scofield Sajan George Sharon Clay Radhey S. Kaushik Artur J. M. Rosa David Francis Volker S. Br?zel 《Applied and environmental microbiology》2009,75(17):5489-5495
Antibiotics such as chlortetracycline (CTC) have been used to promote growth of pigs for decades, but concerns over increased antibiotic-resistant infections in humans have prompted the development of alternative strategies. Developing alternatives to antibiotic growth promoters (AGPs) could be informed by information on the mechanisms of growth promotion, notably, how AGPs affect the microbial populations of the gastrointestinal tract. Pigs from three sows were aseptically delivered by cesarean section. Six piglets were distributed to each of two foster mothers until weaning, when piglets were fed a diet with or without 50 mg/kg CTC for 2 weeks. The ileal bacterial microbiota was characterized by using a cultivation-independent approach based on DNA extraction, PCR amplification, cloning, and sequencing of the 16S rRNA gene pool. The ileal and mucosal communities of these growing pigs were dominated by Lactobacillus bacteria, various members of the family Clostridiaceae, and members of the poorly known genus Turicibacter. Overall, CTC treatment resulted in three shifts: a decrease in Lactobacillus johnsonii, an increase in L. amylovorus, and a decrease in Turicibacter phylotypes. The composition of the microbiota varied considerably between individual pigs, as revealed by shared operational taxonomic units (OTUs) and similarity (SONS) analysis (θYC values). While the observed variation between untreated pigs obscured the possible effect of CTC, ∫-LIBSHUFF and SONS analyses of pooled libraries indicated a significant shift due to CTC in both the lumen and the mucosa, with some OTUs unique to either treated or control ileum. DOTUR analysis revealed little overlap between control and treated communities at the 3% difference level, indicating unique ileal communities in the presence of CTC.Antibiotics have been used to promote animal growth for over 50 years. Antibiotic growth promoters (AGPs) such as tylosin, bacitracin, virginiamycin, and chlortetracycline (CTC) have been fed to pigs, chickens, and other animals to promote growth through increased feed intake, weight gain, and improved herd health (7, 36). Use of AGPs has come under increasing pressure with the growing consensus that their use leads to increased antibiotic-resistant infections in humans via generation of reservoirs of antibiotic-resistant bacteria that may enter the food chain through contamination (38, 46). The increasing concerns about antibiotic resistance have raised questions about whether the potential risks are worth the beneficial effects (44). Development of non-antibiotic-based alternative strategies to promote animal growth may benefit through increased understanding of AGP mechanisms of growth promotion.The growth-promoting impact of antibiotics was first described in the 1940s, and their use soon became routine (29, 35). The gastrointestinal (GI) tract harbors a great diversity of bacteria at a very high density (27). The increased growth and feed efficiency promoted by AGPs may be due to alteration of the microbiota of the GI tract. Early hypotheses focused on the suppression of pathogenic bacteria (19), but the broad-spectrum antibiotics used as growth promoters do not target specific species. Suggested mechanisms of action have included suppression of subclinical infections, a decrease in the levels of growth-depressing bacterial metabolites, decreased consumption of nutrients by intestinal microbiota, and improvement of nutrient uptake due to a thinner intestinal wall (14, 48). Data on the effect of AGPs on pig intestinal microbiota are needed in order to determine the relative contributions of the various proposed mechanisms. Much of the evidence available points to the action of antibiotics on intestinal bacteria as the main component responsible for the growth effect on animals (17, 20, 36).Traditional culture methods have provided some insights into pig GI microbiota, but culture-independent techniques utilizing analysis of rRNA genes have revealed a far greater diversity. Culture-independent methods have also helped to further our understanding of bacterial population dynamics and the complex interplay between the host and pathogenic and nonpathogenic bacteria. The construction of a large 16S rRNA bacterial clone library from the pig GI tract identified 375 phylotypes by using a similarity criterion of 97% (27). Studies utilizing denaturing gradient gel electrophoresis have shown the microbial variances between compartments of the pig intestinal tract, the effect of the diet on microbial communities of the colon, and the ileal microbiota changes produced by the use of several types of AGP (5, 28, 45). Each technique can hold its own bias or limitation, but combinations of fingerprinting and PCR techniques have led to a greater understanding of the composition of pig GI microbiota and their ecology (16, 49, 50).Studies on the effect of antibiotics on intestinal microbiology have focused on colonic or fecal microbiota because bacterial densities are highest (14) and sampling is noninvasive, allowing temporal studies. Yet, nutrient uptake occurs primarily in the small intestine, the region where bacterial activity would therefore have the greatest influence on growth (14). Demands on the GI tract to respond to bacteria by increased mucus production occur primarily in the small intestine (13). The main growth-promoting effect of antibiotics is therefore more likely to occur in the small intestine, specifically in the ileum, where bacterial numbers have reached a high density. One study showed that AGPs, including bacitracin, CTC, and tylosin, caused a shift in the ileal microbial profile of pigs (5). In that study, only one pig was used per treatment, so the basal variation in microbiota between individuals was not taken into account.The objective of this study was to examine how the AGP CTC affects the microbial community of the porcine ileum. To account for variation in the intestinal microbiota as influenced by both antenatal and postnatal environment, pigs from three separate sows were aseptically delivered by cesarean (C) section and distributed to two foster mothers until weaning, when piglets were fed a diet either with or without the AGP CTC. A cultivation-independent approach based on DNA extraction, PCR amplification, and cloning and sequencing of the 16S RNA gene was taken to characterize the pig ileal microbiota. 相似文献
993.
Denise A. Berti Cain Morano Lilian C. Russo Leandro M. Castro Fernanda M. Cunha Xin Zhang Juan Sironi Cl��cio F. Klitzke Emer S. Ferro Lloyd D. Fricker 《The Journal of biological chemistry》2009,284(21):14105-14116
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme
that has been proposed to metabolize peptides within cells, thereby affecting
antigen presentation and G protein-coupled receptor signal transduction.
However, only a small number of intracellular substrates of EP24.15 have been
reported previously. Here we have identified over 100 peptides in human
embryonic kidney 293 (HEK293) cells that are derived from intracellular
proteins; many but not all of these peptides are substrates or products of
EP24.15. First, cellular peptides were extracted from HEK293 cells and
incubated in vitro with purified EP24.15. Then the peptides were
labeled with isotopic tags and analyzed by mass spectrometry to obtain
quantitative data on the extent of cleavage. A related series of experiments
tested the effect of overexpression of EP24.15 on the cellular levels of
peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10
of the cellular peptides were incubated with purified EP24.15 in
vitro, and the cleavage was monitored by high pressure liquid
chromatography and mass spectrometry. Many of the EP24.15 substrates
identified by these approaches are 9–11 amino acids in length,
supporting the proposal that EP24.15 can function in the degradation of
peptides that could be used for antigen presentation. However, EP24.15 also
converts some peptides into products that are 8–10 amino acids, thus
contributing to the formation of peptides for antigen presentation. In
addition, the intracellular peptides described here are potential candidates
to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and
if this process is impaired, the elevated levels of aged proteins usually lead
to the formation of intracellular insoluble aggregates that can cause severe
pathologies (1). In mammalian
cells, most proteins destined for degradation are initially tagged with a
polyubiquitin chain in an energy-dependent process and then digested to small
peptides by the 26 S proteasome, a large proteolytic complex involved in the
regulation of cell division, gene expression, and other key processes
(2,
3). In eukaryotes, 30–90%
of newly synthesized proteins may be degraded by proteasomes within minutes of
synthesis (3,
4). In addition to proteasomes,
other extralysosomal proteolytic systems have been reported
(5,
6). The proteasome cleaves
proteins into peptides that are typically 2–20 amino acids in length
(7). In most cases, these
peptides are thought to be rapidly hydrolyzed into amino acids by
aminopeptidases
(8–10).
However, some intracellular peptides escape complete degradation and are
imported into the endoplasmic reticulum where they associate with major
histocompatibility complex class I
(MHC-I)3 molecules and
traffic to the cell surface for presentation to the immune system
(10–12).
Additionally, based on the fact that free peptides added to the intracellular
milieu can regulate cellular functions mediated by protein interactions such
as gene regulation, metabolism, cell signaling, and protein targeting
(13,
14), intracellular peptides
generated by proteasomes that escape degradation have been suggested to play a
role in regulating protein interactions
(15). Indeed, oligopeptides
isolated from rat brain tissue using the catalytically inactive EP24.15 (EC
3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and
were found capable of altering G protein-coupled receptor signal transduction
(16). Moreover, EP24.15
overexpression itself changed both angiotensin II and isoproterenol signal
transduction, suggesting a physiological function for its intracellular
substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family
that contains the HEXXH motif
(17). This enzyme was first
described as a neuropeptide-degrading enzyme present in the soluble fraction
of brain homogenates (18).
Whereas EP24.15 can be secreted
(19,
20), its predominant location
in the cytosol and nucleus suggests that the primary function of this enzyme
is not the extracellular degradation of neuropeptides and hormones
(21,
22). EP24.15 was shown in
vivo to participate in antigen presentation through MHC-I
(23–25)
and in vitro to bind
(26) or degrade
(27) some MHC-I associated
peptides. EP24.15 has also been shown in vitro to degrade peptides
containing 5–17 amino acids produced after proteasome digestion of
β-casein (28). EP24.15
shows substrate size restriction to peptides containing from 5 to 17 amino
acids because of its catalytic center that is located in a deep channel
(29). Despite the size
restriction, EP24.15 has a broad substrate specificity
(30), probably because a
significant portion of the enzyme-binding site is lined with potentially
flexible loops that allow reorganization of the active site following
substrate binding (29).
Recently, it has also been suggested that certain substrates may be cleaved by
an open form of EP24.15 (31).
This characteristic is supported by the ability of EP24.15 to accommodate
different amino acid residues at subsites S4 to S3′, which even includes
the uncommon post-proline cleavage
(30). Such biochemical and
structural features make EP24.15 a versatile enzyme to degrade structurally
unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15
were isolated and identified using mass spectrometry
(22). The majority of peptides
captured by the inactive enzyme were intracellular protein fragments that
efficiently interacted with EP24.15; the smallest peptide isolated in these
assays contained 5 and the largest 17 amino acids
(15,
16,
22,
32), which is within the size
range previously reported for natural and synthetic substrates of EP24.15
(18,
30,
33,
34). Interestingly, the
peptides released by the proteasome are in the same size range of EP24.15
competitive inhibitors/substrates
(7,
35,
36). Taken altogether, these
data suggest that in the intracellular environment EP24.15 could further
cleave proteasome-generated peptides unrelated to MHC-I antigen presentation
(15).Although the mutated inactive enzyme “capture” assay was
successful in identifying several cellular protein fragments that were
substrates for EP24.15, it also found some interacting peptides that were not
substrates. In this study, we used several approaches to directly screen for
cellular peptides that were cleaved by EP24.15. The first approach involved
the extraction of cellular peptides from the HEK293 cell line, incubation
in vitro with purified EP24.15, labeling with isotopic tags, and
analysis by mass spectrometry to obtain quantitative data on the extent of
cleavage. The second approach examined the effect of EP24.15 overexpression on
the cellular levels of peptides in the HEK293 cell line. The third set of
experiments tested synthetic peptides with purified EP24.15 in vitro,
and examined cleavage by high pressure liquid chromatography and mass
spectrometry. Collectively, these studies have identified a large number of
intracellular peptides, including those that likely represent the endogenous
substrates and products of EP24.15, and this original information contributes
to a better understanding of the function of this enzyme in vivo. 相似文献
994.
Imke E Mulder Bettina Schmidt Christopher R Stokes Marie Lewis Mick Bailey Rustam I Aminov James I Prosser Bhupinder P Gill John R Pluske Claus-Dieter Mayer Corran C Musk Denise Kelly 《BMC biology》2009,7(1):1-20
Background
Early microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. Recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. The extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. We addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development.Results
Genetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. Analysis of over 3,000 16S rRNA sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in early-life environment. Pigs housed in a natural outdoor environment showed a dominance of Firmicutes, in particular Lactobacillus, whereas animals housed in a hygienic indoor environment had reduced Lactobacillus and higher numbers of potentially pathogenic phylotypes. Our analysis revealed a strong negative correlation between the abundance of Firmicutes and pathogenic bacterial populations in the gut. These differences were exaggerated in animals housed in experimental isolators. Affymetrix microarray technology and Real-time Polymerase Chain Reaction revealed significant gut-specific gene responses also related to early-life environment. Significantly, indoor-housed pigs displayed increased expression of Type 1 interferon genes, Major Histocompatibility Complex class I and several chemokines. Gene Ontology and pathway analysis further confirmed these results.Conclusion
Early-life environment significantly affects both microbial composition of the adult gut and mucosal innate immune function. We observed that a microbiota dominated by lactobacilli may function to maintain mucosal immune homeostasis and limit pathogen colonization. 相似文献995.
Marla K. Johnson Tamara D. Clark Denise Njama-Meya Philip J. Rosenthal Sunil Parikh 《PloS one》2009,4(9)
Background
Clinical association studies have yielded varied results regarding the impact of glucose-6-phosphate dehydrogenase (G6PD) deficiency upon susceptibility to malaria. Analyses have been complicated by varied methods used to diagnose G6PD deficiency.Methodology/Prinicipal Findings
We compared the association between uncomplicated malaria incidence and G6PD deficiency in a cohort of 601 Ugandan children using two different diagnostic methods, enzyme activity and G6PD genotype (G202A, the predominant East African allele). Although roughly the same percentage of males were identified as deficient using enzyme activity (12%) and genotype (14%), nearly 30% of males who were enzymatically deficient were wild-type at G202A. The number of deficient females was three-fold higher with assessment by genotype (21%) compared to enzyme activity (7%). Heterozygous females accounted for the majority (46/54) of children with a mutant genotype but normal enzyme activity. G6PD deficiency, as determined by G6PD enzyme activity, conferred a 52% (relative risk [RR] 0.48, 95% CI 0.31–0.75) reduced risk of uncomplicated malaria in females. In contrast, when G6PD deficiency was defined based on genotype, the protective association for females was no longer seen (RR = 0.99, 95% CI 0.70–1.39). Notably, restricting the analysis to those females who were both genotypically and enzymatically deficient, the association of deficiency and protection from uncomplicated malaria was again demonstrated in females, but not in males (RR = 0.57, 95% CI 0.37–0.88 for females).Conclusions/Significance
This study underscores the impact that the method of identifying G6PD deficient individuals has upon association studies of G6PD deficiency and uncomplicated malaria. We found that G6PD-deficient females were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency is described using enzyme activity. These observations may help to explain the discrepancy in some published association studies involving G6PD deficiency and uncomplicated malaria. 相似文献996.
c-Myc interacts with components of the pre-replication complex and directly regulates DNA replication [1]. However the consequences of this novel c-Myc function on cell cycle dynamics and replication-associated damage are unknown. Here, we show that c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase while c-Myc deficient fibroblasts exhibit a prolonged S-phase. We also show that the Werner DNA helicase protein (WRN) plays a critical role in supporting c-Myc-driven S-phase, as depletion of WRN in c-Myc overexpressing cells increases DNA damage specifically at sites of DNA synthesis. This excess DNA damage activates a “replication stress” pathway involving ATR, CHK1, CHK2, and p53, leading to rapid senescence of WRN deficient c-Myc overexpressing cells. Indeed, depletion of p53 rescues this senescence response. We propose that WRN functions to repair abnormal replication structures caused by the acceleration of DNA replication by c-Myc. This work provides an additional mechanistic explanation for c-Myc-induced DNA damage and senescence, and reveals a vulnerability of c-Myc overexpressing cells that could potentially be exploited in cancer therapy. 相似文献
997.
Shun Liang Nuo Yang Yue Pan Shan Deng Xiaojuan Lin Xiaojun Yang Dionyssios Katsaros Katherine F. Roby Thomas C. Hamilton Denise C. Connolly George Coukos Lin Zhang 《PloS one》2009,4(1)
Background
The Phosphatidylinositol 3′-kinase is a key regulator in various cancer-associated signal transduction pathways. Genetic alterations of its catalytic subunit alpha, PIK3CA, have been identified in ovarian cancer. Our in vivo data suggests that PIK3CA activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear.Methodology/Principal Findings
Using the Müllerian inhibiting substance type II receptor (MISIIR) promoter, we generated transgenic mice that expressed activated PIK3CA in the Müllerian epithelium. Overexpression of PIK3CA in OSE induced remarkable hyperplasia, but was not able to malignantly transform OSE in vivo. The consistent result was also observed in primary cultured OSEs. Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.Conclusions/Significance
While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras. 相似文献998.
Paola Sebastiani Monty Montano Annibale Puca Nadia Solovieff Toshio Kojima Meng C. Wang Efthymia Melista Micah Meltzer Sylvia E. J. Fischer Stacy Andersen Stephen H. Hartley Amanda Sedgewick Yasumichi Arai Aviv Bergman Nir Barzilai Dellara F. Terry Alberto Riva Chiara Viviani Anselmi Alberto Malovini Aya Kitamoto Motoji Sawabe Tomio Arai Yasuyuki Gondo Martin H. Steinberg Nobuyoshi Hirose Gil Atzmon Gary Ruvkun Clinton T. Baldwin Thomas T. Perls 《PloS one》2009,4(12)
Background
The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered.Methodology/Principal Findings
Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function.Conclusions/Significance
Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan. 相似文献999.