Mangan-superoksid-dismutaza (MnSOD) katalizuje NO-zavisno nitrovanje ostatka tirozina

The peroxynitrite-induced nitration of manganese superoxide dismutase (MnSOD) tyrosine residue, which causes enzyme inactivation, is well established, This led to suggestions that MnSOD nitration and inactivation in vivo, detected in various diseases associated with oxidative stress and overproduction of nitric monoxide (NO), conditions which favor peroxynitrite formation, is also caused by peroxynitrite. However, our previous ill vitro study demonstrated that exposure of MnSOD to NO led to NO conversion into nitrosonium (NO+) and nitroxyl (NO-) species, which caused enzyme modifications and inactivation. Here it is reported that MnSOD is tyrosine nitrated upon exposure to NO, as well as that MnSOD nitration contributes to inactivation of the enzyme. Collectively, these observations provide a compelling argument supporting the generation of nitrating species in MnSOD exposed to NO and shed a new light on MnSOD tyrosine nitration and inactivation ill vivo. This may represent a novel mechanism by which MnSOD protects cell from deleterious effects associated with overproduction of NO. However, extensive MnSOD modification and inactivation associated with prolonged exposure to NO will amplify the toxic effects caused by increased cell superoxide and NO levels.Dobro je poznato da peroksinitrit izaziva nitrovanje ostataka tirozina u mangan-superoksid- dismutazi (MnSOD) što dovodi do inaktivacije enzima. Pokazano je da nitrovanje i inaktivacija MnSOD-a nastaje u raznim bolestima za koje je karakteristič an oksidativni stres i povećana produkcija azot-monoksida (NO). Pošto se pri ovim uslovima očekuje nastajanje peroksinitrita predloženo je da peroksinitrit izaziva nitrovanje i inaktivaciju MnSOD in vivo. U našem prethodnom radu pokazali smo da MnSOD katalizuje transformaciju NO u nitrozonijum (NO+) i nitroksil (NO–) reaktivne vrste, te identifikovali neke od modifikacija molekula enzima koje pri tome nastaju izazivajući njegovu inaktivaciju. U ovom radu je pokazano da pri izlaganju MnSOD azot-monoksidu dolazi i do nitrovanja ostatka tirozina u molekulu enzima, što doprinosi njegovoj inaktivaciji. Ovi rezultati ukazuju da pri interakciji MnSOD sa NO dolazi do nastajanja nitrujućih vrsta, što baca novo svetlo na proces nitrovanja ostataka tirozina i inaktivaciju MnSOD in vivo. Ovo može da predstavlja novi mehanizam kojim MnSOD štiti ćeliju odštetnih efekata izazvanih hiperprodukcijom azot-monoksida. Međutim ekstenzivne modifikacije i inaktivacija MnSOD do kojih dolazi pri produženom izlaganju enzima NO, uvećaće toksične efekte izazvane povećanim koncentracijama superoksida i NO u ćeliji

Performance analysis of a reduced form-factor high accuracy three-axis teslameter

In the framework of the SwissFEL project at the Paul Scherrer Institute (PSI), a Hall probe bench is being developed for the high-precision magnetic characterization of the insertion devices for the ATHOS soft X-ray beamline. For this purpose, a novel three-axis teslameter has been developed, which will be placed between the undulator and its outer shell in a very limited volumetric space of 150 × 50 × 45 mm. Together with a SENIS® 3-axis Hall probe at the center of the cross sectional area of the undulator, the setup will traverse along the undulator length on a specifically designed rig with minimal vibrations. This teslameter has all the analog signal conditioning circuitry for the Hall probe and also has on board 24-bit digitization. The instrument also handles an interface to a linear absolute encoder. The old instrumentation used only had analog signal conditioning circuitry whilst digitization was done off board. The new instrument also provides a very accurate magnetic field map in the µT range with simultaneous readings from the position encoder at an accuracy of ±3 µm. In this paper, a series of tests are described, which were performed at PSI in order to establish the measuring precision and repeatability of the instrument.peer-reviewe

Single-molecule imaging for unraveling the functional diversity of 10–23 DNAzymes

DNA-based enzymes, also known as DNAzymes, have opened new opportunities for signal generation and amplification in several fields including biosensing. However, biosensor performance can be hampered by heterogeneity in the catalytic activity of such DNAzymes, especially when relying on a limited number of molecules to generate signal. In this regard, single-molecule studies are essential to discern the behavior among such heterogeneous molecules otherwise masked by ensemble measurements. This work presents a novel methodology to study the 10–23 RNA-cleaving DNAzyme at the single-molecule level. By means of measuring the distance-sensitive efficiency of Förster Resonance Energy Transfer using alternating-laser excitation on a superresolution microscope, we determined the kinetics of individual DNAzymes in terms of substrate turnover, rates of different reaction steps, and changes in performance over time. Our results revealed that, despite high concentrations of the reaction cofactor (i.e., Mg2+), a maximum of only 70% of the DNAzymes are actively cleaving multiple substrate sequences; the DNAzyme molecules also showed a wide range of substrate turnover rates. Our findings shed new light on the functional diversity of DNAzymes and the importance of exploring sequence modifications to improve their catalytic performance. Ultimately, this work presents a technique to obtain time-dependent information, which could be easily implemented to study other types of enzymes or biomolecular interactions

Rer1p competes with APH-1 for binding to nicastrin and regulates γ-secretase complex assembly in the early secretory pathway

The γ-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this “proteasome of the membrane” are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of γ-secretase subcomplexes and, concomitantly, total cellular γ-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates γ-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular γ-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value

Study of the growth traits relationship of lambs in the postnatal development

Data from the Pirot improved sheep were use to estimate postnatal development and growth traits relationship of lambs from birth to weaning. The experiment included 360 lambs, divided into three groups (I, II, III). Lamb traits included BW at birth and approximately 30d, 60d and 90d (weaning). Lambs managed under conditions typical of the area. Male lambs in-group I had a total gain of 22.97 kg (0.255 kg/d), in group II 25.97 kg (0.286 kg/d) (P (lt) 0.01). Lambs in-group III the total gain was 24.64 kg (0.274 kg/d), which was lower than lambs of group II (P>0.05). On the other side, III group of lambs had a higher gain than the I group (P (lt) 0.01). Development of female lambs in the postnatal period was slightly weaker. Lambs of I group, from birth to weaning had a total gain of 21.27 kg, (0.236 kg/d), II group was 23.32 kg (0.259 kg/d). The difference was statistically very significant (P (lt) 0.01). Lambs of group III had a total gain of 23.54 kg (0.261kg/d) and higher growth rate then lambs of group II, but not significant (P>0.05). From the other side, the difference in comparison with the groups III and I was very significant (P (lt) 0.01). Correlations between BWB and BW30, 60, 90 are ranged from low to moderate among the respective traits and ranged between positive from 0.001 to 0.365 and negative from -0.005 to -0.279. Can conclude that the selection should direct towards producing lambs with intermediate birth weight

Comparison of linear type traits of daughters of Simmental bulls

Linear assessment of the type and body development provides very important information on the total value of animals for the purpose of breeding animals selected and classified into classes. Determination of the average values of exterior parameters, especially non-measurable traits, is useful in defining breeding objectives and formulating effective plans and programs. Given that previous studies found statistically significant differences among daughters of Simmental bulls for some properties of the exterior, the objective of this paper was to compare linear type traits and body development of daughters of Simmental bulls in the Rasina region. Based on linear type traits and body development, the most significant results were achieved by daughters of bull No. 1464 (Ralfus) for the frame, muscularity and form, and by daughters of bull No. 1488 (Hormaz) for udder appearance. If each attribute is analyzed separately, bull No. 1411 (Horau) is equally significant for the traits for which its daughters showed the best performance.Publishe

Da li holesterol vezan za hemoglobin utiče na anti-oksidativni enzimski sistem u humanim eritrocitima?

In a previous study, it was shown that the lipid fraction, which is occasionally observed in red blood cell hemolysates, represents cholesterol (Ch) associated with phospholipid firmly bound to haemoglobin (termed Hb-Ch). The current study was conducted to investigate whether Hb-Ch could affect the primary anti-oxidant enzyme defence system in human erythrocytes. Sixty healthy volunteers were used for the current study. Group 1 consisted of 28 subjects without or with a low level of Hb-Ch. Group 2 comprised 32 subjects with a considerably higher level of Hb-Ch. The activities of erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as the content of methaemoglobin (metHb) were measured in both groups. The results indicated that the amount of Hb-Ch neither influenced the activities of the erythrocyte anti-oxidant enzymes nor altered the level of metHb. However, a higher amount of Hb-Ch changed the correlations in the part of the anti-oxidant defence system relating to glutathione, suggesting increased peroxidative pressure from plasma lipids. Group 2 also had significantly increased concentrations of total plasma Ch and triglycerides. Together, these facts are strong indications that the anti-oxidant defence system in human erythrocytes finely retunes its composition according to plasma oxidative demands.U prethodnom radu pokazano je da lipidna frakcija koja se javlja u hemolizatu zdravih ljudi predstavlja holesterol (asosovan sa fosfolipidima) čvrsto vezan za hemoglobin (Hb-Ch). U ovom radu ispitivan je uticaj Hb-Ch na anti-oksidativni enzimski sistem u humanim eritrocitima. Određena je aktivnost superoksid-dizmutaze, katalaze, glutation-peroksidaze i glutation-reduktaze, kao i sadržaj met-hemoglobina (metHb) u eritrocitima 60 ljudi, podeljenih u dve grupe na osnovu količine Hb-Ch. Rezultati pokazuju da količina prisutnog Hb-Ch ne menja aktivnost merenih enzima, niti nivo metHb. Međutim, u grupi ispitanika sa povećanim sadržajem Hb-Ch zapažene su korelativne promene u delu anti-oksidativnog enzimskog sistema povezanog sa glutationom. U istoj grupi detektovane su i veće koncentracije ukupnog holesterola i triglicerida u plazmi, što zajedno ukazuje na povećani peroksidativni pritisak iz plazme. Ovi rezultati ukazuju da odbrambeni anti-oksidativni enzimski sistem u humanim eritrocitima prilagođava svoju organizaciju prema zahtevima iz svog okruženja.

FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices

Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well-characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO-SPR) bioassay. In this context, EV binding on the FO-SPR probes was achieved only with EV-specific antibodies (e.g. anti-CD9 and anti-CD63) but not with non-specific anti-IgG. To increase detection sensitivity, we tested six different combinations of EV-specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti-CD9/(B)anti-CD81 and anti-CD63/(B)anti-CD9), resulting in 10(3) and 10(4) times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti-CD63/(B)anti-CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti-EpCA M antibody on the FO-SPR surface. The obtained results combined with FO-SPR real-time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis

Continuous Biosensing to Monitor Acute Systemic Inflammation, a Diagnostic Need for Therapeutic Guidance

Continuous monitoring of acute inflammation can become a very important next step for guiding therapeutic interventions in severely ill patients. This Perspective discusses the current medical need for patients with acute inflammatory diseases and the potential of continuous biosensing technologies. First, we discuss biomarkers that could help to monitor the state of a patient with acute systemic inflammation based on theoretical studies and empirical data. Then, based on the state of the art, we describe sensing strategies that could be applied for the continuous monitoring of acute inflammatory biomarkers, followed by challenges that must be overcome. Nanoswitch-based continuous biosensors enable suitable measurement frequencies but still lack sensitivity, while regeneration risks lower sensor reliability. Developments are still needed in bioreceptors and molecular architectures, regeneration techniques, combined with suitable sampling and sample pretreatment methods, for bringing continuous biosensing of inflammation closer to reality. Furthermore, collaborations between healthcare professionals and scientists, regulatory bodies, and biosensor engineers are needed for a successful translation of sensing technologies from the laboratory to clinical practice.</p