The protein synthesis machinery of the cell, the ribosome and associated factors, is able to accurately follow the canonical genetic code, that which maps RNA sequence to protein sequence, to ...assemble functional proteins from the twenty or so proteinogenic amino acids. A number of innovative methods have arisen to take advantage of this accurate, and efficient, machinery to direct the assembly of non-proteinogenic amino acids. We review and compare these routes to 'reprogram the genetic code' including in vitro translation, engineered aminoacyl tRNA synthetases, and RNA 'flexizymes'. These studies show that the ribosome is highly tolerant of unnatural amino acids, with hundreds of unusual substrates of varying structure and chemistries being incorporated into protein chains. We also discuss how these methods have been coupled to selection techniques, such as phage display and mRNA display, opening up an exciting new avenue for the production of proteins and peptides with properties and functions beyond that which is possible using proteins composed entirely of the proteinogenic amino acids.
Objective
To examine the association of dietary habits with high total antioxidant capacity (TAC) with frailty among elderly Japanese women.
Design
Cross-sectional multicenter study.
Setting
...Thirty-five of 47 prefectures in Japan.
Participants
2121 grandmothers or acquaintances of dietetic students aged 65 and older.
Measurements
Dietary TAC and food intakes were calculated using a validated brief-type self-administered diet history questionnaire. The TAC value of each food was assigned using four different assays, ferric reducing ability of plasma (FRAP), oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), and total radical-trapping antioxidant parameter (TRAP). Frailty was defined as the presence three or more of the following four components: slowness and weakness (two points), exhaustion, low physical activity, and unintentional weight loss.
Results
The number of subjects with frailty was 486 (23%). Multivariate adjusted ORs (95% CI) for frailty in the highest compared to the lowest quintile were 0.35 (0.24, 0.53) for FRAP, 0.35 (0.23, 0.52) for ORAC, 0.40 (0.27, 0.60) for TEAC, and 0.41 (0.28, 0.62) for TRAP. The intakes of green tea, coffee, vegetables, and fruits which contribute to dietary TAC were also associated with lower odds of frailty (the range of multivariate adjusted OR: 0.47 for vegetables to 0.77 for green tea), although the odds ratios were less marked than those of dietary TAC.
Conclusions
Dietary habits with high TAC showed a stronger inverse association with frailty in elderly Japanese women than the individual foods examined.
-Methylated amino acids (
-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However,
discovery from ribosome display is limited by poor incorporation of
...-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the
-methyl-aminoacyl-tRNA. By reconfiguring the tRNA's T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different
-MeAAs. We have here devised a "pool-and-split" enrichment strategy using the RaPID display and identified
-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57%
-methylation with up to three consecutively incorporated
-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by
-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two
-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent
iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the
-MeAA MCP library.
Objectives The “tissue oxygen saturation (StO2 ) foot-mapping” method was developed using a non-invasive near-infrared tissue oximeter monitor to classify the foot regions as ischemic and ...non-ischemic areas. The purpose of this study was to evaluate StO2 foot-mapping as a reliable method to detect ischemic areas in the feet of patients with critical limb ischemia (CLI), and to compare the results with assessments from the angiosome model. Methods The foot areas of 20 CLI patients and 20 healthy controls were classified into four regions: (1) 0 ≤ StO2 < 30%, (2) 30 ≤ StO2 < 50%, (3) 50 ≤ StO2 < 70%, and (4) 70 ≤ StO2 ≤ 100% to perform StO2 foot-mapping. Each area occupancy rate was compared between the two groups, and the threshold StO2 value for detecting ischemia was set. Next, the locations of ulcers (in 16 patients) were compared to the predicted ischemic regions by the StO2 foot-mapping and by the angiosome model and angiography. Results In regions (1) and (2) (StO2 < 50%), the area occupancy rate was significantly higher in the CLI group and almost zero in the control group, so that the threshold StO2 value for detecting ischemia was set at 50%. The locations of ulcers were compatible with StO2 foot-mapping in 87.5% of the cases (14/16), while they were compatible with the assessment from the angiosome model in 68.8% of the cases (11/16). Conclusions This study suggests that StO2 foot-mapping can successfully and non-invasively detect ischemic areas in the peripheral tissue of the foot, and also more appropriately than the assessment provided by the angiosome model. StO2 foot-mapping can be used to evaluate the real angiosome: the real distribution of the peripheral tissue perfusion in the CLI patient's foot, which is determined by the peripheral microvascular blood flow, rather than the main arterial blood flow.
Tumor cells in cutaneous T-cell lymphoma express limited numbers of chemokine receptors. We investigated the expression patterns of CXCR3, CCR3, CCR4 and CCR10 in mycosis fungoides, Sézary syndrome, ...lym-phomatoid papulosis and anaplastic large cell lymphoma in 121 skin biopsy samples. CXCR3 was expressed in 86% of mycosis fungoides cases but in no anaplastic large cell lymphoma cases. CCR3 was expressed in 73% of cases of CD30+ lymphoproliferative disorders such as lymphomatoid papulosis and anaplastic large cell lymphoma. Mycosis fungoides/Sézary syndrome patients with high CCR3 or CCR4 expression had a poorer survival prognosis than mycosis fungoides/Sézary syndrome patients whose tumor cells did not express these receptors. CCR10 was expressed in 50% of mycosis fungoides/Sézary syndrome cases and in 13% of cases with CD30+ lym-phoproliferative disorders. These results suggest that differential patterns of CXCR3, CCR3, CCR4 and CCR10 expression are useful for the diagnosis of cutaneous T-cell lymphoma. Moreover, expression of CCR3 or CCR4 suggests a poor prognosis in mycosis fungoides/Sézary syndrome.
Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. ...graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.
Summary
Background
Interleukin (IL)‐25 is a member of the IL‐17 family, which can promote and augment T‐helper (Th) type 2 responses. The expression of IL‐25 and its cognate receptor, IL‐25 receptor ...(IL‐25R), is upregulated and correlated with disease activity in Th2‐associated diseases.
Objectives
To examine the expression and function of IL‐25 in cutaneous T‐cell lymphoma (CTCL).
Methods
Expression and location of IL‐25 in lesional skin was investigated with immunohistochemistry. The effect of various cytokines on IL‐25 production from normal human epidermal keratinocytes was assessed by quantitative reverse‐transcription real‐time polymerase chain reaction. Serum IL‐25 levels were measured by enzyme‐linked immunosorbent assay. The direct effect of IL‐25 on tumour cells was also examined using CTCL cell lines and peripheral blood mononuclear cells in patients with Sézary syndrome.
Results
IL‐25 expression was increased in epidermal keratinocytes in lesional skin of CTCL. Th2 cytokines, IL‐4 and IL‐13, and periostin induced IL‐25 expression by normal human epidermal keratinocytes. Serum IL‐25 levels were increased in patients with advanced CTCL and correlated with serum lactate dehydrogenase levels. MyLa cells expressed IL‐25R and its expression was augmented by stimulation with IL‐25. IL‐25 enhanced IL‐13 production from MyLa cells via phosphorylation of signal transducer and activator of transcription 6. Peripheral blood mononuclear cells from one patient with Sézary syndrome expressed IL‐25R and showed increase of IL‐13 production by IL‐25.
Conclusions
Th2 cytokines highly expressed in CTCL lesional skin induce IL‐25 production by epidermal keratinocytes, which may, in turn, lead to formation of a Th2‐dominant microenvironment through the direct induction of IL‐13 by tumour cells.
What's already known about this topic?
Cutaneous T‐cell lymphomas (CTCLs), such as mycosis fungoides and Sézary syndrome, are regarded as T helper 2 (Th2)‐type diseases, and a Th2‐dominant microenvironment is beneficial for tumour cells.
Interleukin (IL)‐25 has the capacity to promote and augment Th2 responses and is associated with several Th2‐type diseases, including atopic dermatitis.
What does this study add?
Th2 cytokines highly expressed in lesional skin of CTCL induce IL‐25 production by epidermal keratinocytes.
IL‐25 directly induces IL‐13 production from CTCL tumour cells through signal transducer and activator of transcription 6 (STAT6) signalling pathways, resulting in the formation of a Th2‐dominant microenvironment.
What is the translational message?
Our results support the notion that activation of STAT6 is a key signalling pathway for the creation of a Th2‐dominant microenvironment in CTCL.
As the destruction of a Th2‐dominant microenvironment is effective for CTCL, IL‐25 and STAT6 can be a therapeutic target for CTCL.
We report a strategy for efficient post-translational modification of a library of ribosomally-translated peptides by activation and elimination of cysteine to dehydroalanine then conjugate addition ...of a range of exogenous thiols, with an emphasis on carbohydrates. These reactions are selective for cysteine, and do not interfere with amplification of the nucleic acid component of an mRNA-displayed peptide. Furthermore, these reactions are shown to be compatible with two different macrocyclisation chemistries, and when applied to a peptide containing an
-terminal cysteine give a ketone that can be functionalised in an orthogonal manner. This new strategy can overcome a limitation of ribosomal translation, providing a means to incorporate untranslatable groups such as carbohydrates in amino acid side chains, and will allow for the ribosomal generation of glycopeptides, requiring only the introduction of a free thiol in the molecule to be incorporated. In combination with
selection techniques, this strategy is envisaged to allow the discovery of biologically-active glycopeptides with a near-natural, but hydrolytically stable, thioglycosidic bond.
Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA ...display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.
Fusarium fujikuroi
is the pathogen of rice
bakanae
disease and has been intensively studied for gibberellin (GA) production.
F. fujikuroi
is phylogenetically subclassified into G- and F-groups, which ...differ in GA and fumonisin production. A higher amount of GA is produced by the G-group than the F-group. A previous study found that the GA production difference between the G-group strain Gfc0801001 and the F-group strain Gfc0825009 was due to the GA gene cluster. In this study, higher expression of
P450–1
and
P450–4
in the GA gene cluster were detected in G-group strains than in F-group strains. The nucleotide sequence of the bidirectional promoter region between
P450–1
and
P450–4
and expression of
P450–1
and
P450–4
were compared between G- and F-group strains. The promoter sequence was identical among G-group strains while nucleotide substitutions were detected at various locations in F-group strains. Most F-group strains did not share the same substitutions. These results combined with the ancestral position of the F-group in the phylogenetic tree suggest that the population carrying highly expressed sequence generates the G-group rather than the low expression of
P450–1
and
P450–4
in the F-group due to a specific mutation occured in G-group.
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