Inhibition Of Capping Research Articles

E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

Carbon nanotubes influence the toxic effects of chloramphenicol and tetracycline on cyanobacteriumSynechocystissp. in different ways

CNTs mitigated the inhibition of CAP on protein biosynthesis, while CAP enhanced the upregulation of proteins induced by CNTs. TC can potentiate the CNT-induced downregulation of proteins related to ribosome.

Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin

We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739-751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.

Development and characterization of an allergoid of cat dander for immunotherapy

BackgroundAllergy to cats is a frequent cause of sensitization to indoor allergens and currently there are few alternatives to specific immunotherapy with cat native extracts. The objective is to develop and characterize a new allergoid to increase the tools available for use in clinical practice. MethodsThe allergoid cat dander extract (ACD) was developed from a native cat dander extract (NCD) by modification with glutaraldehyde, and the optimal process control was determined by SDS-PAGE, DOT BLOT and determination of free amine groups. The ACD was characterized in protein profile by SDS-PAGE, size exclusion chromatography (SEC) and peptide footprint. The allergenic profile of ACD was determined by immunoblot, IgE CAP inhibition and IgG competition ELISA. The major allergen content in NCD was obtained by the ELISA sandwich protocol and was extrapolated to ACD. ResultsThe control process determined the optimal development of the allergoid. The ACD obtained contains 182.28μg/mg of protein and 11.90μg/mg of Fel d 1. SDS-PAGE and SEC confirmed the presence of high molecular weight proteins in ACD, and the peptide footprint showed the presence of Fel d 1 and Fel d 7. The high degree of polymerization was evidenced with the determination of the reduction of lysine residues in the allergoid, resulting 91.96%. The ACD showed a significant loss of allergenicity respect to NCD, while the IgG-binding capacity was maintained. ConclusionsThe ACD obtained presents a good safety profile, so would be a good alternative for treatment of cat allergy.

Fruit sensitization in patients with allergy to latex

In the last years, latex has frequently been found to be involved in immediate hypersensitivity reactions. The first case mentioned with recurrent urticaria and laryngoedema was reported by Stern (1) in 1927. Since then, latex has also been implicated in generalized urticaria, rhinoconjunctivitis, asthma and anaphylaxis. Associated sensitization to several fruits is frequently seen in latex-allergic patients with the symptoms described above. This study was performed in seven patients (six females and one male) with hypersensitivity to latex and concomitant fruit sensitization. Six of them were healthcare personnel. The age of the patients ranged from 25-39 years, with a mean of 30 years. Prick tests and intracutaneous tests with latex (10% w/v in PBS), banana, chestnut, avocado, kiwi and melon were carried out. A specific histamine release test (HRT) was performed according to the fluorometric assay. Antigen-specific IgE was also performed. Latex CAP inhibition with banana and SDS-PAGE immunoblotting were carried out in one patient. Although in latex-allergic patients multiple sensitization to fruits may be observed, banana and avocado are those most frequently involved, followed by chestnut and melon. This is likely to be due to the presence of common antigens in these fruits and latex, as demonstrated in our study only for banana and avocado. We consider that further investigation is needed on the possible sensitization to latex in sanitary personnel reporting symptoms after fruit ingestion.

Comparing the ability of molecular diagnosis and CAP-inhibition in identifying the really causative venom in patients with positive tests to Vespula and Polistes species.

BackgroundCross-reactivity among Hymenoptera venoms is an important issue when prescribing venom immunotherapy (VIT). Using all venoms eliciting a positive response results in treatment excess and unjustified cost increase. The first in vitro method that helped to identify the really causative venom was RAST-inhibition, but in latest years also molecular allergy (MA) diagnostics, that detects specific sIgE to single venom allergens, was introduced. We compared the two methods in patients with double sensitization to Vespula spp. and Polistes spp.MethodsFifty-four patients with anaphylactic reactions to Hymenoptera stings and positive results to skin tests and sIgE measurement with whole venom from Vespula spp. and Polistes dominula were included in the study. Sera from all patients were analyzed by CAP-inhibition (Thermo Fisher Scientific, Uppsala, Sweden) and MA diagnostics with recombinant Ves v 1, Ves v 5 and Pol d 5.ResultsBy the data obtained from MA technique, VIT would have been prescribed to 7 patients for Polistes, to 6 for Vespula, and to 41 for both venoms. With the data from CAP inhibition, it would have been a prescription to 15 patients for Polistes, to 28 for Vespula, and to 11 for both venoms. A good concordance between the results of MA and CAP-inhibition was found only when the value in kU/l of Ves v 5 were about twice those of Pol d 5, and vice versa.ConclusionsThese findings suggest that in the choice of the venom to be used for VIT CAP-inhibition remains a pivotal tool, because the significance of in vitro inhibition is definite and provides a diagnostic importance higher than MA in patients with positive tests to both Vespula and Polistes spp.

Content of Asthmagen Natural Rubber Latex Allergens in Commercial Disposable Gloves.

The use of natural rubber latex (NRL) gloves in many occupations may lead to latex sensitization, allergic asthma, and skin reactions. Due to their good properties and environmental safety NRL gloves are still being used in the healthcare setting, but also in the food industry, by hairdressers, cleaners, etc. The aim of our study was to assess the protein and NRL allergen content in commercial gloves by different methods, including a new assay. Twenty commercially available NRL gloves were analyzed. Protein extraction was performed according to the international standard ASTM D-5712. Total protein content was measured with a modified Lowry method, NRL content with the CAP Inhibition Assay, the Beezhold ELISA Inhibition Assay, and an innovative ELISA with IgY-antibodies extracted from eggs of NRL-immunized hens (IgY Inhibition Assay). We found a high protein content in a range of 215.0-1304.7μg/g in 8 out of the 20 NRL gloves. Seven of the 20 gloves were powdered, four of them with a high protein content. In gloves with high protein content, the immunological tests detected congruently high levels of NRL allergen. We conclude that a high percentage of commercially available NRL gloves still represent a risk for NRL allergy, including asthma. The modified Lowry Method allows to infer on the latex allergen content.

Content of Asthmagen Natural Rubber Latex Allergens in Commercial Disposable Gloves.

The use of natural rubber latex (NRL) gloves in many occupations may lead to latex sensitization, allergic asthma, and skin reactions. Due to their good properties and environmental safety NRL gloves are still being used in the healthcare setting, but also in the food industry, by hairdressers, cleaners, etc. The aim of our study was to assess the protein and NRL allergen content in commercial gloves by different methods, including a new assay. Twenty commercially available NRL gloves were analyzed. Protein extraction was performed according to the international standard ASTM D-5712. Total protein content was measured with a modified Lowry method, NRL content with the CAP Inhibition Assay, the Beezhold ELISA Inhibition Assay, and an innovative ELISA with IgY-antibodies extracted from eggs of NRL-immunized hens (IgY Inhibition Assay). We found a high protein content in a range of 215.0-1304.7μg/g in 8 out of the 20 NRL gloves. Seven of the 20 gloves were powdered, four of them with a high protein content. In gloves with high protein content, the immunological tests detected congruently high levels of NRL allergen. We conclude that a high percentage of commercially available NRL gloves still represent a risk for NRL allergy, including asthma. The modified Lowry Method allows to infer on the latex allergen content.

Wasp venom allergy screening with recombinant allergen testing. Diagnostic performance of rPol d 5 and rVes v 5 for differentiating sensitization to Vespula and Polistes subspecies

BackgroundThe appropriate therapeutic management of patients with wasp-induced allergic reactions necessitates an accurate allergologic workup, entailing differentiation between sensitization against Vespula or Polistes venoms. Materials and methodsWe studied 52 consecutive adult subjects diagnosed with hypersensitivity to wasp venoms. Cap inhibition was performed using UniCAP Specific IgE. The concentration of serum IgE against two recombinant constituents of Antigen 5 (rVes v 5 and rPol d 5) was also measured using Immuno CAP 250. The ratio between values of specific IgE against recombinant allergens was calculated and a percentage difference >50% was considered significant for specific immunization against one of the two venoms. ResultsThe diagnostic agreement between recombinant allergens testing and CAP inhibition was 54% (kappa statistics, 0.34; 95% CI, 0.18–0.50) in the whole study population. In the 24 patients with recombinant allergens ratio >50% and non dubious results of CAP inhibition assay the diagnostic agreement was perfect (100%; kappa of agreement, 1.00; 95% CI; 1.00–1.00). DiscussionThe results of this study show that the assessment of specific IgE against rVes v 5 and rPol d 5 may be regarded as a low-cost screening, providing valuable diagnostic information for differentiating the sensitization against Vespula or Polistes venoms. In patients with suggestive clinical history and ratio >50% between specific IgE against rVes v 5 and specific IgE against and rPol d 5, the CAP inhibition assay may be safely withheld, thus allowing to achieve an early diagnosis at lower cost.

Murine norovirus (MNV-1) exposure in vitro to the purine nucleoside analog Ribavirin increases quasispecies diversity

Ribavirin is a pharmaceutical antiviral used for the treatment of RNA virus infections including norovirus, hepatitis C virus, hepatitis E virus, Lassa virus, respiratory syncytial virus, and rhinovirus. Despite the drug's history and documented efficacy, the antiviral mechanism of Ribavirin remains unclear. Mechanisms proposed include depletion of the intracellular GTP pool, immunomodulatory effects, induction of error catastrophe, inhibition of viral polymerase activity, and/or inhibition of viral capping. In the present study, we leveraged deep sequencing data to demonstrate that Ribavirin increases murine norovirus (MNV-1) viral diversity. By serial passaging MNV-1 in RAW 264.7 cells for twenty generations in the presence of Ribavirin, we demonstrated statistically significant increases in both the number of unique haplotypes and the average pairwise difference (APD). Based on statistically significant differences in the probability of nucleotide mutations based on Roche 454 sequencing, we also demonstrated that single nucleotide substitutions are increased in the presence of Ribavirin. Finally, we demonstrated Ribavirin's impact on statistically significantly reducing the relative proportion of the dominant sequence within the quasispecies.

Ligustrazine Suppresses the Growth of HRPC Cells through the Inhibition of Cap- Dependent Translation Via Both the mTOR and the MEK/ERK Pathways.

Ligustrazine (TMP) has recently been used for the treatment of various cancers. However, its exact mechanisms of action, particularly the functions and the mechanisms of Ligustrazine in human hormone-refractory prostate cancer (HRPC), have not yet been extensively studied. Recently, our findings suggest that Ligustrazine dose- and time-dependently inhibits the growth of HRPC cells by reducing their proliferation and promoting apoptosis. Interestingly, the treatment of hormone-refractory prostate cancer (PC-3) cells with Ligustrazine results in a significant inhibition of the activation of mTOR and related downstream targets, which are critical for cell growth. Furthermore, pull-down assays with 7-methyl- GTP Sepharose 4B beads indicate that Ligustrazine reduces the available eIF4E for translation initiation. Accordingly, the results from the translation assay using a luciferase reporter system further demonstrate that Ligustrazine indeed inhibits cap-dependent translation. In addition, the transient overexpression of eIF4E or MNK1 prevents the Ligustrazine-induced inhibition of proliferation and confers significant protection against Ligustrazine-induced apoptosis. Therefore, the present study provides evidences that Ligustrazine may be a candidate for therapeutic reagent for the treatment of HRPC and certifies that Ligustrazine modulates the availability of eIF4E mainly through the mTOR and MEK/ERK signaling pathways to inhibit cap-dependent translation. Taken together, our results indicate that the inhibition of cap-dependent translation is likely an essential mechanism in Ligustrazine-induced apoptosis.

The cytoplasmic capping complex assembles on adapter protein nck1 bound to the proline-rich C-terminus of Mammalian capping enzyme.

Author SummaryWe previously described a cyclical process of mRNA decapping and recapping termed “cap homeostasis.” Recapping is catalyzed by a complex of cytoplasmic proteins that includes the enzyme known to catalyze nuclear capping, and a kinase that converts RNA with a 5′-monophosphate end to a 5′-diphosphate capping substrate. The current study shows these two enzymatic activities are brought together in the cytoplasmic capping complex as both bind to adjacent domains of the adapter protein Nck1. Nck1 is a cytoplasmic protein best known for transducing receptor tyrosine kinase signaling. We identify a proline-rich sequence at the C-terminus of a human capping enzyme that is required for binding to Nck1, and we show that this interaction is required for integrity of the cytoplasmic capping complex. Depletion of Nck1 causes the cytoplasmic capping complex to dissociate. The inhibition of cytoplasmic capping by Nck1 with mutations in either the 5′-kinase or capping enzyme binding sites identified a functional role for Nck1 in cap homeostasis and a previously unknown function for Nck1 in cell biology.

Isolated IgE reactivity to native walnut vicilin-like protein (nJug r 2) on ISAC™ microarray is due to cross-reactive carbohydrate epitopes

The last version of the microarray-based testing ImmunoCAP ISAC 112™ includes the native walnut (Junglans regia) molecules 2S albumin (nJug r 1), vicilin (nJug r 2) and lipid transfer protein (nJug r 3). In view of the many unexpected cases of isolated positivity to nJug r 2 occurring in daily practice, we evaluated the association of these reactivities with clinical symptoms, as well as the relationship between sIgE and nJug r 2 and cross-reactive carbohydrate determinants (CCDs). Sera from 320 consecutive allergic outpatients tested by ImmuoCAP ISAC™ 112 were considered. The medical records of all nJug r 2 positive patients were reviewed to assess clinical symptoms related to walnut allergy. A linear regression analysis was performed to evaluate the correlation between nJug r 2 and CCDs (nMUXF3) sIgE values, and a CAP inhibition assay was carried out to confirm the possible cross-reactivity between CCDs and nJug r 2. Thirty-seven out of 320 sera tested (11.6%) were positive to nJug r 2. Among them three (8.1%) and eight (21.6%) scored positive for nJug r 1 and nJug r 3 as well, respectively. Twenty-seven (73%) sera showed isolated nJug r 2 positivity. Only nJug r 1 reactors had symptoms referred to walnut allergy. Twenty-five/37 nJug r 2-positive sera (67.6%) showed a simultaneous positivity to nMUXF3 and a significant correlation (p<0.0001) between the IgE levels to nJug r 2 and nMUXF3 (r²=0.787). After incubation with nMUXF3 a complete inhibition of sIgE reactivity to both nMUXF3 and nJug r 2 was shown. The unexpected isolated sIgE reactivity to nJug r 2 found by ImmunoCAP ISAC™ 112 is frequently related to reactivity to cross-reactive carbohydrate epitopes and it is lacking clinical significance.

Protease nexin 1: a novel regulator of prostate cancer cell growth and neo-angiogenesis

Prostate adenocarcinoma (CaP) is the most frequent cancer and second leading cause of cancer death among men in the Western world [1]. Hh signaling may augment the development of CaP as emerging data suggests that components of the pathway are potential indicators of worse prognosis [2]. One major clinical difficulty, however, is that many prostate cancers are indolent and cause no difficulty while others are aggressive and lead to morbidity and mortality. Current methods to distinguish CaP types are inadequate and could lead to overtreatment in many cases. We recently have shown that genetic alteration in the Hedgehog (Hh) pathway impacts CaP growth experimentally and may correlate with clinical outcome [3]. Examination of a cohort of intermediate risk prostate carcinoma patients by comparative genomic hybridization (CGH) for a panel of hedgehog related genes indicated a more rapid progression to PSA (prostate antigen) failure, bolstering this argument. Thus, studying the pathway may reveal promising avenues for distinguishing outcomes and identifying new targets for therapy. Our work has shown that Hh signaling can be regulated in part by a protein normally expressed in the prostate, protease nexin 1 (PN1, also known as serpinE2) [3]. PN1 is a serine protease inhibitor (serpin) with the ability to bind and neutralize the activity of thrombin, trypsin, and urokinase (uPA) [4]. The regulation of uPA is meaningful in cancer because of its role in the cleavage and activation of plasminogen to plasmin, which in turn activates matrix metalloproteinases (MMPs) and ECM remodelling [5]. Previously, we established that PN1-mediated inhibition of uPA served to inhibit metastatic prostate cell invasion in Matrigel [6]. Our new study features several components. Firstly, we show that PN1 inhibits Hh signaling by reduction in the hedgehog ligand, Sonic (SHH). This regulation is dependent upon uptake of PN1 through the LRP receptor and on the inhibitory activity of PN1. The immediate functional impact of PN1 is to reduce proliferation of prostate metastatic cells both in culture and in vivo. Indeed, inducing expression of PN1 in several different prostate cancer cells was as effective in the inhibition of proliferation as treatment with the Hh-pathway inhibitor cyclopamine. Hh signaling inhibition also substantially reduced neo-angiogenesis. Xenografts from PC3 prostate cancer cells expressed high levels of SHH, producing small, disorganized vessels typical of tumour vasculature. These results are in line with other reports linking the Hedgehog pathway with the formation of new blood vessels and increases in tumor growth and metastasis [7]. By contrast, tumors treated with hedgehog inhibitors like GDC-0449 and pre-treated with PN1 recombinant protein exhibited altered vasculature with fewer vessels and larger overall diameters. The consequence of reduction in hedgehog signaling was decreased overall vascular density and slower tumor growth. The capacity to reduce tumor growth and vessel formation suggests a new physiological significance for PN1 and perhaps other serpins with similar functions. Interestingly, PN1 features some similarities to maspin, a tumor suppressor and angiogenesis blocker. Maspin has also been reported to reduce uPA-mediated CaP [8]. However, PN1 is more stable in a purified recombinant state than maspin and may actually have a larger range of inhibitory targets or cell type interactions [8]. For example, a recent report has observed that PN1 may be able to interact with endothelial cells, the initiators of angiogenesis [9]. As a regulatory toggle, we have shown that PN1 is also a target of MMP9-mediated degradation. The localisation of PN1 in the ECM allows for its regulation by MMP9, an important factor in metastasis that can cleave collagen and basement membrane to drive this process. We extended these findings to show that higher MMP9 levels are inverse to PN1 levels and that this ratio is important in controlling hedgehog signaling. We demonstrate competing regulation of hedgehog signaling by PN1 and MMP9 in tissue culture, normal prostate tissues, xenografts, and orthotopic prostate cancers. The presence of PN1 in normal and BPH (benign prostate hypertrophy) tissues and decline in higher Gleason score samples may indicate that its loss is a marker of malignant transition in the prostate epithelium, amplifying Hh signaling. Indeed, one aim was to utilize Hh signalling as a stratifier of intermediate grade prostate cancer as the transitional status of these patients often makes treatment uncertain. Implications are that pharmacological inhibition of hedgehog signaling might be useful in this subset of patients. Thus, PN1 itself might be a potential therapeutic agent and, in combination with other SHH inhibitors, may be useful in the encouragement of CaP growth lag or inhibition. This work further indicates the potential important of hedgehog signaling in prostate cancer (Figure ​(Figure1).1). Given its emerging abilities to affect changes in proliferation, angiogenesis, and invasion, PN1 expression may be a suitable target for improving prostate cancer therapy. Figure 1 Expression of SHH in prostate cancer

Identification of Cytoplasmic Capping Targets Reveals a Role for Cap Homeostasis in Translation and mRNA Stability

The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5' ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3' untranslated regions of recapping targets are enriched for AU-rich elements and microRNA binding sites, both of which function in cap-dependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.

Programmed Cell Death 4 (PDCD4): A Novel Player in Ethanol‐Mediated Suppression of Protein Translation in Primary Cortical Neurons and Developing Cerebral Cortex

Prenatal exposure to ethanol (EtOH) elicits a range of neuro-developmental abnormalities, microcephaly to behavioral deficits. Impaired protein synthesis has been connected to pathogenesis of EtOH-induced brain damage and abnormal neuron development. However, mechanisms underlying these impairments of protein synthesis are not known. In this study, we illustrate the effects of EtOH on programmed cell death protein 4 (PDCD4), a tumor and translation repressor. Primary cortical neurons (PCNs) were treated with 2.5 and 4 mg/ml EtOH for different time points (4 to 24 hours), and PDCD4 expression was detected by Western blotting. Protein synthesis was determined using [(35) S] methionine incorporation assay. Methyl cap pull-down assay was performed to establish the effect of EtOH on association of eukaryotic initiation factor 4A (eIF4A) with capped mRNA. Luciferase assay was performed to determine the in vivo translation. A 2-day acute 5-dose binge model with EtOH (4 g/kg body wt, 25% v/v) was performed in Sprague-Dawley rats at 12-hour intervals and analyzed for PDCD4, eIF4A, and eIF4A-methyl cap association. EtOH increased PDCD4 expression in a time- and dose-dependent manner in PCNs, which inhibited the association of eIF4A with methyl cap. EtOH and ectopic PDCD4 expression suppressed in vivo translation in PCNs and RNAi targeting of PDCD4 blocked the inhibitory effect of EtOH on protein synthesis. In utero exposure of pregnant rats to EtOH resulted in a significant increase in PDCD4 in fetal cerebral cortex along with the inhibition of methyl cap-associated eIF4A, compared with isocaloric controls. Increased PDCD4 also occurred in pooled fractions of remaining brain regions. Our data, for the first time, illustrate that PDCD4 mediates inhibitory effects of EtOH on protein synthesis in PCNs and developing brain.

Methyl Jasmonate Induces ATP Biosynthesis Deficiency and Accumulation of Proteins Related to Secondary Metabolism in Catharanthus roseus (L.) G. Hairy Roots

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.

Glutaraldehyde-Modified Recombinant Fel d 1: A Hypoallergen With Negligible Biological Activity but Retained Immunogenicity

Background Recombinant allergens are under investigation for replacing allergen extracts in immunotherapy. Site-directed mutagenesis has been suggested as a strategy to develop hypoallergenic molecules that will reduce the risk of side effects. For decades, chemically modified allergen extracts have been used for the same reason.Aim To evaluate whether glutaraldehyde modification is a good strategy to produce hypoallergenic recombinant allergens with retained immunogenicity.Methods Fel d 1 was cloned as a single construct linking both chains of the molecule and expressed in Escherichia coli and Pichia pastoris. After physicochemical purification, recombinant (r) Fel d 1 was chemically modified using glutaraldehyde. The effect of modification on immune reactivity was evaluated using radioallergosorbent test, CAP inhibition, competitive radioimmunoassay, enzyme-linked immunosorbent assay, basophil histamine release, and T-cell proliferation assays. Both natural and recombinant unmodified Fel d 1 were used as controls.Results rFel d 1 demonstrated similar IgE binding and biologic activity as its natural counterpart. Upon modification, IgE-binding potency decreased >1000-fold, translating into a >106-fold reduction in biologic activity assessed by basophil histamine release. In contrast, the modified recombinant did not show a decreased but even a moderately increased capacity (1.5-fold) to stimulate proliferation of T cells (P < 0.01). Finally, it induced specific IgG antibodies in rabbits that recognized the unmodified allergen.Conclusions Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity.

The Interaction of Capping Protein with the Barbed End of the Actin Filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.

Impact of Dermatophagoides pteronyssinus mite body raw material on house dust mite allergy diagnosis in a Serbian population

House dust mite (HDM) allergy has different clinical and immunological patterns in different geographic regions. The impact of raw material of commercial Dermatophadoides pteronyssinus (Acari: Pyroglyphidae) mite bodies on the quality of allergen extracts for allergy diagnosis in the Serbian population has not been previously evaluated. House dust mite bodies obtained from manufacturers in Europe, South America and Australia were used in the preparation of allergen extracts for in vivo diagnosis and serological analysis in a group of 14 HDM-allergic adults. In the group of mite-allergic patients, there was no statistically significant difference in skin test reactivity (Wilcoxon matched pairs test) among the three HDM body extract preparations. In a CAP inhibition assay, two extracts (A and C) achieved maximum inhibition of >90%, whereas extract B demonstrated a different inhibition slope and lower inhibition potential (80%). However, a remarkable difference in immunoglobulin E reactivity using Western blot analysis with individual patients' sera was observed in one of the preparations (extract B). These findings emphasize the need for the careful selection of starting material for the preparation of HDM diagnostic reagents intended for use in patients from geographically distinct regions as these preparations can have implications on the selection criteria for patient-tailored immunotherapy of HDM allergy.