Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.

Urinary incontinence and vaginal squeeze pressure two years post-cesarean delivery in primiparous women with previous gestational diabetes mellitus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Adhesive Bonding of Resin Composite to Various Ni-Cr Alloy Surfaces Using Different Metal Conditioners and a Surface Modification System

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Finite element analysis and fracture resistance testing of a new intraradicular post

Objectives: The objective of the present study was to evaluate a prefabricated intraradicular threaded pure titanium post, designed and developed at the Sao Jose dos Campos School of Dentistry - UNESP, Brazil. This new post was designed to minimize stresses observed with prefabricated post systems and to improve cost-benefits. Materials and and methods: Fracture resistance testing of the post/core/root complex, fracture analysis by microscopy and stress analysis by the finite element method were used for post evaluation. The following four prefabricated metal post systems were analyzed: group 1, experimental post; group 2, modification of the experimental post; group 3, Flexi Post, and group 4, Para Post. For the analysis of fracture resistance, 40 bovine teeth were randomly assigned to the four groups (n=10) and used for the fabrication of test specimens simulating the situation in the mouth. The test specimens were subjected to compressive strength testing until fracture in an EMIC universal testing machine. After fracture of the test specimens, their roots were sectioned and analyzed by microscopy. For the finite element method, specimens of the fracture resistance test were simulated by computer modeling to determine the stress distribution pattern in the post systems studied. Results: The fracture test presented the following averages and standard deviation: G1 (45.63 +/- 8.77), G2 (49.98 +/- 7.08), G3 (43.84 +/- 5.52), G4 (47.61 +/- 7.23). Stress was homogenously distributed along the body of the intraradicular post in group 1, whereas high stress concentrations in certain regions were observed in the other groups. These stress concentrations in the body of the post induced the same stress concentration in root dentin. Conclusions: The experimental post (original and modified versions) presented similar fracture resistance and better results in the stress analysis when compared with the commercial post systems tested (08/2008PA/CEP).

Sequencing wasp venom peptides by endopeptidase digestion and nested collision-induced dissociation/post-source decay methods

A method incorporating nested collision-induced dissociation/post-source decay (CID/PSD) combined with endopeptidase digestion is described as an approach to determine the sequence of N-terminally modified peptides. The information from immonium and related ions observed in the CID/PSD spectrum was used for the selection of a suitable endopeptidase for the digestion of peptides. Rapid and reliable assignment of peptide sequence was performed by the comparison of CID/PSD spectra of both intact and endopeptidese-digested peptide fragments, since the assignments of the observed fragment ions to either N- or C-terminal ions can thus be carried out unambiguously. This nested CID/PSD method was applied to the sequence determination of two peptides from the solitary wasps Anoplius samariensis and Batozonellus maculifrons (pompilid wasps), which could not be sequenced by the Edman method due to N-terminal modification. Copyright (C) 2002 John Wiley Sons, Ltd.

Adhesive bonding of resin composite to various titanium surfaces using diferente metal conditioners and a surface modification system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of active recombinant eIF5A: reconstitution in E. coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.

Engineering the synthesis of lantibiotics in e. Coli by combining the cynnamicin and nisin modification systems.

I lantibiotici sono molecole peptidiche prodotte da un gran numero di batteri Gram-positivi, posseggono attività antibatterica contro un ampio spettro di germi, e rappresentano una potenziale soluzione alla crescente problematica dei patogeni multi-resistenti. La loro attività consiste nel legame alla membrana del bersaglio, che viene quindi destabilizzata mediante l’induzione di pori che determinano la morte del patogeno. Tipicamente i lantibiotici sono formati da un “leader-peptide” e da un “core-peptide”. Il primo è necessario per il riconoscimento della molecola da parte di enzimi che effettuano modifiche post-traduzionali del secondo - che sarà la regione con attività battericida una volta scissa dal “leader-peptide”. Le modifiche post-traduzionali anticipate determinano il contenuto di amminoacidi lantionina (Lan) e metil-lantionina (MeLan), caratterizzati dalla presenza di ponti-tioetere che conferiscono maggior resistenza contro le proteasi, e permettono di aggirare la principale limitazione all’uso dei peptidi in ambito terapeutico. La nisina è il lantibiotico più studiato e caratterizzato, prodotto dal batterio L. lactis che è stato utilizzato per oltre venti anni nell’industria alimentare. La nisina è un peptide lungo 34 amminoacidi, che contiene anelli di lantionina e metil-lantionina, introdotti dall’azione degli enzimi nisB e nisC, mentre il taglio del “leader-peptide” è svolto dall’enzima nisP. Questo elaborato affronta l’ingegnerizzazione della sintesi e della modifica di lantibiotici nel batterio E.coli. In particolare si affronta l’implementazione dell’espressione eterologa in E.coli del lantibiotico cinnamicina, prodotto in natura dal batterio Streptomyces cinnamoneus. Questo particolare lantibiotico, lungo diciannove amminoacidi dopo il taglio del leader, subisce modifiche da parte dell’enzima CinM, responsabile dell’introduzione degli aminoacidi Lan e MeLan, dell’enzima CinX responsabile dell’idrossilazione dell’acido aspartico (Asp), e infine dell’enzima cinorf7 deputato all’introduzione del ponte di lisinoalanina (Lal). Una volta confermata l’attività della cinnamicina e di conseguenza quella dell’enzima CinM, si è deciso di tentare la modifica della nisina da parte di CinM. A tal proposito è stato necessario progettare un gene sintetico che codifica nisina con un leader chimerico, formato cioè dalla fusione del leader della cinnamicina e del leader della nisina. Il prodotto finale, dopo il taglio del leader da parte di nisP, è una nisina completamente modificata. Questo risultato ne permette però la modifica utilizzando un solo enzima invece di due, riducendo il carico metabolico sul batterio che la produce, e inoltre apre la strada all’utilizzo di CinM per la modifica di altri lantibiotici seguendo lo stesso approccio, nonché all’introduzione del ponte di lisinoalanina, in quanto l’enzima cinorf7 necessita della presenza di CinM per svolgere la sua funzione.

Association of biomarkers of lipid modification with functional and morphological indices of coronary stenosis severity in stable coronary artery disease

Biomarkers of blood lipid modification and oxidative stress have been associated with increased cardiovascular morbidity. We sought to determine whether these biomarkers were related to functional indices of stenosis severity among patients with stable coronary artery disease. We studied 197 consecutive patients with stable coronary artery disease due to single vessel disease. Fractional flow reserve (FFR) ≤ 0.80 was assessed as index of a functionally significant lesion. Serum levels of secretory phospholipase A2 (sPLA2) activity, secretory phospholipase A2 type IIA (sPLA2-IIA), myeloperoxydase (MPO), lipoprotein-associated phospholipase A2 (Lp-PLA2), and oxidized low-density lipoprotein (OxLDL) were assessed using commercially available assays. Patients with FFR > 0.8 had higher sPLA2 activity, sPLA2 IIA, and OxLDL levels than patients with FFR ≤ 0.8 (21.25 [16.03-27.28] vs 25.85 [20.58-34.63] U/mL, p < 0.001, 2.0 [1.5-3.4] vs 2.6 [2.0-3.4] ng/mL, p < 0.01; and 53.0 [36.0-71.0] vs 64.5 [50-89.25], p < 0.001 respectively). Patients with FFR > 0.80 had similar Lp-PLA2 and MPO levels versus those with FFR ≤ 0.8. sPLA2 activity, sPLA2 IIA significantly increased area under the curve over baseline characteristics to predict FFR ≤ 0.8 (0.67 to 0.77 (95 % confidence interval [CI]: 0.69-0.85) p < 0.01 and 0.67 to 0.77 (95 % CI: 0.69-0.84) p < 0.01, respectively). Serum sPLA2 activity as well as sPLA2-IIA level is related to functional characteristics of coronary stenoses in patients with stable coronary artery disease.

Internet-based interpretation bias modification for social anxiety: A pilot study

BACKGROUND AND OBJECTIVES: The biased interpretation of ambiguous social situations is considered a maintaining factor of Social Anxiety Disorder (SAD). Studies on the modification of interpretation bias have shown promising results in laboratory settings. The present study aims at pilot-testing an Internet-based training that targets interpretation and judgmental bias. METHOD: Thirty-nine individuals meeting diagnostic criteria for SAD participated in an 8-week, unguided program. Participants were presented with ambiguous social situations, were asked to choose between neutral, positive, and negative interpretations, and were required to evaluate costs of potential negative outcomes. Participants received elaborate automated feedback on their interpretations and judgments. RESULTS: There was a pre-to-post-reduction of the targeted cognitive processing biases (d = 0.57-0.77) and of social anxiety symptoms (d = 0.87). Furthermore, results showed changes in depression and general psychopathology (d = 0.47-0.75). Decreases in cognitive biases and symptom changes did not correlate. The results held stable accounting for drop-outs (26%) and over a 6-week follow-up period. Forty-five percent of the completer sample showed clinical significant change and almost half of the participants (48%) no longer met diagnostic criteria for SAD. LIMITATIONS: As the study lacks a control group, results lend only preliminary support to the efficacy of the intervention. Furthermore, the mechanism of change remained unclear. CONCLUSION: First results promise a beneficial effect of the program for SAD patients. The treatment proved to be feasible and acceptable. Future research should evaluate the intervention in a randomized-controlled setting.

An assessment of post-disaster psychological stress in hazardous waste operations and emergency response (HAZWOPER) workers

Personnel involved in natural or man-made disaster response and recovery efforts may be exposed to a wide variety of physical and mental stressors that can exhibit long-lasting and detrimental psychopathological outcomes. In a disaster situation, huge numbers of "secondary" responders can be involved in contaminant clean-up and debris removal and can be at risk of developing stress-related mental health outcomes. The Occupational Safety and Health Administration (OSHA) worker training hierarchy typically required for response workers, known as "Hazardous Waste Operations and Emergency Response" (HAZWOPER), does not address the mental health and safety concerns of workers. This study focused on the prevalence of traumatic stress experienced by secondary responders that had received or expressed interest in receiving HAZWOPER training through the National Institute of Environmental Health Sciences Worker Education and Training Program (NIEHS WETP). ^ The study involved the modification of two preexisting and validated survey tools to assess secondary responder awareness of physical, mental, and traumatic stressors on mental health and sought to determine if a need existed to include traumatic stress-related mental health education in the current HAZWOPER training regimen. The study evaluated post-traumatic stress disorder (PTSD), resiliency, mental distress, and negative effects within a secondary responder population of 176 respondents. Elevated PTSD levels were seen in the study population as compared to a general responder population (32.9% positive vs. 8%-22.5% positive). Results indicated that HAZWOPER-trained disaster responders were likely to test positive for PTSD, whereas, untrained responders with no disaster experience and responders who possessed either training or disaster experience only were likely to test PTSD negative. A majority (68.75%) of the population tested below the mean resiliency to cope score (80.4) of the average worker population. Results indicated that those who were trained only or who possessed both training and disaster work experience were more likely to have lower resiliency scores than those with no training or experience. There were direct correlations between being PTSD positive and having worked at a disaster site and experiencing mental distress and negative effects. However, HAZWOPER training status does not significantly correlate with mental distress or negative effect. ^ The survey indicated clear support (91% of respondents) for mental health education. The development of a pre- and post-deployment training module is recommended. Such training could provide responders with the necessary knowledge and skills to recognize the symptomology of PTSD, mental stressors, and physical and traumatic stressors, thus empowering them to employ protective strategies or seek professional help if needed. It is further recommended that pre-deployment mental health education be included in the current HAZWOPER 24- and 40-hour course curriculums, as well as, consideration be given towards integrating a stand-alone post-deployment mental health education training course into the current HAZWOPER hierarchy.^

Post-transcriptional Regulation of Mammalian Gene Expression in Non-coding Region of Target RNA

Tumor Suppressor Candidate 2 (TUSC2) is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. TUSC2 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous TUSC2 protein could be detected in a majority of lung cancer cell lines. Mechanisms regulating TUSC2 protein expression and its inactivation in primary lung cancer cells are largely unknown. We investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the TUSC2 gene in the regulation of TUSC2 protein expression. We found that two small upstream open-reading frames (uORFs) in the 5’UTR of TUSC2 could markedly inhibit the translational initiation of TUSC2 protein by interfering with the “scanning” of the ribosome initiation complexes. Site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) verified several micoRNAs (miRNAs) targeted at 3’UTR and directed TUSC2 cleavage and degradation. In addition, we used the established let-7-targeted high mobility group A2 (Hmga2) mRNA as a model system to study the mechanism of regulation of target mRNA by miRNAs in mammalian cells under physiological conditions. There have been no evidence of direct link between mRNA downregulation and mRNA cleavages mediated by miRNAs. Here we showed that the endonucleolytic cleavages on mRNAs were initiated by mammalian miRNA in seed pairing style. Let-7 directed cleavage activities among the eight predicted potential target sites have varied efficiency, which are influenced by the positional and the structural contexts in the UTR. The 5’ cleaved RNA fragments were mostly oligouridylated at their 3’-termini and accumulated for delayed 5’–3’ degradation. RNA fragment oligouridylation played important roles in marking RNA fragments for delayed bulk degradation and in converting RNA degradation mode from 3’–5’ to 5’–3’ with cooperative efforts from both endonucleolytic and non-catalytic miRNA-induced silencing complex (miRISC). Our findings point to a mammalian miRNA-mediated mechanism for the regulation of mRNA that miRNA can decrease target mRNA through target mRNA cleavage and uridine addition

Quantum key distribution based on selective post-processing in passive optical networks

One of the main obstacles to the widespread adoption of quantum cryptography has been the difficulty of integration into standard optical networks, largely due to the tremendous difference in power of classical signals compared with the single quantum used for quantum key distribution. This makes the technology expensive and hard to deploy. In this letter, we show an easy and straightforward integration method of quantum cryptography into optical access networks. In particular, we analyze how a quantum key distribution system can be seamlessly integrated in a standard access network based on the passive optical and time division multiplexing paradigms. The novelty of this proposal is based on the selective post-processing that allows for the distillation of secret keys avoiding the noise produced by other network users. Importantly, the proposal does not require the modification of the quantum or classical hardware specifications neither the use of any synchronization mechanism between the network and quantum cryptography devices.

Polymorphism in the 3′-untranslated region of TNFα mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFα mRNA

Tumor necrosis factor α (TNFα) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3′-untranslated region (3′-UTR) of TNFα mRNA as this region plays a pivotal role in post-transcriptional control of TNFα production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFα mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFα. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3′-UTR of TNFα mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFα protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFα 3′-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFα mRNA, these results suggest that mutations in the ARE of TNFα mRNA decrease the production of TNFα protein in macrophages by hindering the binding of HuR to the ARE.