Literatura académica sobre el tema ""zero-length" dimer"

Synapsin I is believed to play an important role in the regulation of neurotransmitter release, since it is able to bind to synaptic vesicles, to the cytoskeleton and to membrane proteins; in addition, it bundles F-actin and microtubules. These properties, which are controlled by phosphorylation, could be explained if synapsin has different and multiple binding sites or if synapsin I is able to form polymers by self-association. In this study we present experimental evidence that synapsin I at low concentration forms self-associated dimers, as revealed after mild treatments with cross-linking agents. We have especially studied here the effects of copper/o-phenanthroline, a zero-length cross-linking agent which forms covalent links by oxidative formation of S-S bridges between adjacent cysteines. The time course and concentration-dependence of synapsin-dimer formation are studied; interestingly, these experiments could suggest a different behaviour of the two polypeptides. Limited proteolysis of phosphorylated synapsin I by V8 protease, alpha-chymotrypsin or collagenase, performed on the isolated dimer and monomer, allows us to localize tentatively in the central hydrophobic core of the molecule the cysteine residues the oxidation of which by copper/o-phenanthroline gives rise to synapsin dimers.

Abstract Factor VIIIa is a heterotrimer of A1, A2, and A3-C1-C2 subunits, the activity of which is labile due to a weak affinity interaction of the A2 subunit with the A1/A3-C1-C2 dimer. We have used the zero-length cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), to localize regions of interaction within the A1 and A2 subunits. Reaction of factor VIIIa with EDC resulted in the formation of a cross-linked product of approximately 90 kD consisting of the A1 and A2 subunits as judged by Western blotting. Alkaline resistance of this product indicated an amide rather than ester linkage. Factor VIIIa activity decreased as the concentration of cross-linked product increased, suggesting that flexibility in the inter-subunit interaction may be required for proper cofactor function. This product was not formed in the contiguous A1-A2 domains of factor VIII, suggesting that, upon cofactor activation, a conformational change occurs that leads to the formation of a new interdomainal salt bridge(s). Reaction of the EDC-treated factor VIIIa with activated protein C (APC), which cleaves the A1 subunit at Arg336 and bisects the A2 subunit at Arg562, resulted in the formation of an approximately 30 kD product that contains the C-terminus region of A1 covalently linked to the N-terminal half of the A2. The approximately 90 kD cross-linked product was generated after reaction of A2 subunit with A1/A3-C1-C2 dimer but not with A1336/A3-C1-C2, a form of the dimer produced by APC cleavage and lacking the C-terminal acidic region of A1. A synthetic peptide corresponding to this acidic region (Met337-Arg372) was found to covalently cross-link to the isolated A2 subunit in 1:1 stoichiometry, suggesting that this region is both necessary and sufficient for the interaction of the A1 and A2 subunits. Sequence analysis of this product suggested that Glu344 in the A1 peptide may contribute to the cross-linkage. These results indicate that activation of factor VIII results in formation of a new ionic linkage(s) localized to the acidic C-terminal region of A1 and the N-terminal half of A2.

Introduction: A patient's response to sepsis is influenced by their genetic background. Our objective was to use plasma markers, such as protein C (PC), D-dimer, Plasminogen Activator Inhibitor-1 (PAI-1) levels, and the PAI-1 rs1799889 4G/5G and Tumor Necrosis Factor-α rs1800629 G/A polymorphisms to improve classical intensive care unit (ICU) scores. Methodology: We studied 380 subjects, 166 with sepsis. We performed coagulation tests: plasma PAI-1 and PC levels were evaluated by chromogenic methods; and D-dimer was evaluated by immunoturbidimetric assay. Polymorphisms were performed using for polymerase chain reactions followed by digest with specific restriction enzyme. We acquired the APACHE and SOFA scores (time zero), sex, age, body mass index, associated co-morbidities, length of ICU stay (days), the severity of sepsis (sepsis, severe sepsis or septic shock), the HIV status and the ICU outcome (survival or death). Results: We found significant differences between patients who died (n=80) and those who survived (n=86) in terms of the ICU length of stay (6 vs. 10 days), septic shock (64 versus 24%), age (51 versus 38 years old), HIV+ condition (34 versus 16%), SOFA (7 versus 4), APACHE (19 versus 13), D-dimer (4.32 versus 2.88 mg/ml), PC (46.0 versus 63.5 %) and PAI-1 (33.0 versus 16.5 UA/l). When we used a regression analysis with dichotomized variables, only the SOFA4, PAI-116, HIV status and the PAI-1 4G allele proved to be predictors of death at time zero. Conclusions: In the future, ICU scores may be further improved by adding certain genomic or plasma data.

Recombinant DNA technology has been used to analyze the first step in keratin intermediate filament (IF) assembly; i.e., the formation of the double stranded coiled coil. Keratins 8 and 18, lacking cysteine, were subjected to site specific in vitro mutagenesis to change one amino acid in the same relative position of the alpha-helical rod domain of both keratins to a cysteine. The mutations lie at position -36 of the rod in a "d" position of the heptad repeat pattern, and thus air oxidation can introduce a zero-length cystine cross-link. Mutant keratins 8 and 18 purified separately from Escherichia coli readily formed cystine homodimers in 2 M guanidine-HCl, and could be separated from the monomers by gel filtration. Heterodimers with a cystine cross-link were obtained when filaments formed by the two reduced monomers were allowed to oxidize. Subsequent ion exchange chromatography in 8.5 M urea showed that only a single dimer species had formed. Diagonal electrophoresis and reverse phase HPLC identified the dimer as the cystine containing heterodimer. This heterodimer readily assembled again into IF indistinguishable from those obtained from the nonmutant counterparts or from authentic keratins. In contrast, the mixture of cystine-stabilized homodimers formed only large aberrant aggregates. However, when a reducing agent was added, filaments formed again and yielded the heterodimer after oxidation. Thus, the obligatory heteropolymer step in keratin IF assembly seems to occur preferentially at the dimer level and not during tetramer formation. Our results also suggest that keratin I and II homodimers, once formed, are at least in 2 M guanidine-HCl a metastable species as their mixtures convert spontaneously into heterodimers unless the homodimers are stabilized by the cystine cross-link. This previously unexpected property of homodimers explains major discrepancies in the literature on the keratin dimer.

SummaryWe have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

Aim Development of a novel scale for assessing medical state in patients with new coronavirus infection based on clinical and laboratory disease severity's markers, named SHOKS-COVID scale.Material and Methods Clinical Assessment Scale (SHOKS-COVID) is based on1: clinical parameters (respiratory rate, Body temperature, SpO2 need and type of ventilation support) 2: Inflammation markers (C reactive protein (CRP) and prothrombotic marker (D-dimer)) and 3: percent of lungs injury by CT. This scale was used in several clinical studies in patients with varying severity of the course of the COVID 19. SHOKS-COVID scale was also compared against some additional biomarkers and with length of hospital stay.Results In patients with severe COVID-19 (Clinical Trial WAYFARER - 34 patients), SHOKS-COVID scores were correlated with the degree of inflammation: CRP (r = 0.64; p <0.0001); the ratio lymphocytes / CRP (r = - 0.64; p <0.0001). Also, SHOKS-COVID score correlated with the D-dimer (r = 0.35; p <0.0001) and percentage lung damage on multispiral computed tomography (MSCT) - (r = 0.77, p < 0.0001) and length stay in the clinic (r = 0.57, p = 0.0009). In patients with mild course (BISQUIT Study - 103 patients), SHOKS-COVID scores had a statistically significant positive correlation with length of fever (r = 0.37; p = 0.0002) and length of stay in the clinic (r = 0.52, p <0.0001) and negatively correlated with the ratio of lymphocytes / CRP (-0.78, p <0.0001) and the level of CRP (r=0.78; p <0.0001). Patents were grouped based on severity of COVID 19 and median and interquartile range (IQR) of SHOCKS-COVID were measured in these groups. Median and IQR of SHOCKS-COVID were 2.00 [1.0-2.5] points in mild course, 4.0 points [3.0-5.0] in moderate course, 7.0 points [6.0-9.0] in moderately severe course,12.0 points [10.0-14.0] in severe course of disease and 15.0 points [14.5-15.5] in extremely severe patients.Conclusion Here we report a novel scale of COVID 19 disease progression. This scale ranges from zero in asymptomatic patients (with normal range of biomarkers and without lung damage on CT) to fifteen in extremely severe patients. The scores for SHOKS-COVID are increasing, in parallel with the deterioration of all other biomarkers of severity and prognosis in patients with new coronavirus infection. Based on the analysis carried out, we were able to determine values of SHOKS-COVID scale and levels of main clinical and laboratory markers in patients with different severity of COVID 19.

Spectrin assembles into an anti-parallel heterodimeric flexible rod-like molecule through a multistep process initiated by a high affinity interaction between discrete complementary homologous motifs or “repeats” near the actin binding domain. Attempts to determine crystallographic structures of this critical dimer initiation complex have so far been unsuccessful. Therefore, in this study we determined the subunit-subunit docking interface and a plausible medium resolution structure of the heterodimer initiation site using homology modeling coupled with structural refinement based on experimentally determined distance constraints. Intramolecular and intermolecular cross-links formed by the “zero length” cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were identified after trypsin digestion of cross-linked heterodimer complex using liquid chromatography-tandem mass spectrometry analysis. High confidence assignment of cross-linked peptides was facilitated by determination of cross-linked peptide masses with an uncertainty of a few parts per million using a high sensitivity linear ion trap mass spectrometer equipped with a Fouriertransform ion cyclotron resonance detector. Six interchain cross-links distinguished between alternative docking models, and these distance constraints, as well as three intrachain cross-links, were used to further refine an initial homology-based structure. The final model is consistent with all available physical data, including protease protection experiments, isothermal titration calorimetry analyses, and location of a common polymorphism that destabilizes dimerization. This model supports the hypothesis that initial docking of the correct α and β repeats from among many very similar repeats in both subunits is driven primarily by long range electrostatic interactions.

Abstract. We present an innovative instrument telescope, and describe a retrieval method to probe 3-D distributions of atmospheric trace gases that are relevant to air pollution and tropospheric chemistry. The University of Colorado (CU) two dimensional (2-D) Multi-AXis-Differential Optical Absorption Spectroscopy (CU 2D-MAX-DOAS) instrument measures nitrogen dioxide (NO2), formaldehyde (HCHO), glyoxal (CHOCHO), oxygen dimer (O2-O2, or O4) and water vapor (H2O); also nitrous acid (HONO), bromine monoxide (BrO), iodine monoxide (IO) among other gases can in principle be measured. Information about aerosols is derived through coupling with a radiative transfer model (RTM). The 2-D telescope has 3 modes of operation: (mode 1) measures solar scattered photons from any pair of elevation angle (−20° < EA < +90° or zenith; zero is to the horizon) and azimuth angle (−180° < AA < +180°; zero being North), (mode 2) measures any set of AA at constant EA (almucantar scans); and (mode 3) tracks the direct solar beam via a separate view port. Vertical profiles of trace gases are measured, and used to estimate planetary boundary layer height (PBL). Horizontal distributions are then derived using PBL and parameterization of RTM (Sinreich et al., 2013). NO2 is evaluated at different wavelengths (350, 450, and 560 nm), exploiting the fact that the effective path length varies systematically with wavelength. The area probed is constrained by O4 observations at nearby wavelengths, and has an effective radius of 7.5 to 20 km around the instrument location; i.e., up to 1250 km2 can be sampled near-instantaneously, and with high time resolution. The instrument was deployed as part of the Multi Axis DOAS Comparison campaign for Aerosols and Trace gases (MAD-CAT) in Mainz, Germany from 7 June to 6 July 2013. We present first measurements (modes 1 and 2 only) and describe a four-step retrieval to derive (a) boundary layer vertical profiles of NO2 and PBL; (b) near-surface horizontal distributions of NO2; (c) range resolved NO2 horizontal distribution measurements using an "onion peeling" approach; and (d) the ratios HCHO-to-NO2 (RFN), CHOCHO-to-NO2 (RGN), and CHOCHO-to-HCHO (RGF) at 14 pre-set azimuth angles distributed over a 360° view. 2D-MAX-DOAS provides an innovative, regional perspective about trace gases, their spatial and temporal concentration gradients, and maximizes information to compare near-surface observations with atmospheric models and satellites.

Abstract. We present an innovative instrument telescope and describe a retrieval method to probe three-dimensional (3-D) distributions of atmospheric trace gases that are relevant to air pollution and tropospheric chemistry. The University of Colorado (CU) two-dimensional (2-D) multi-axis differential optical absorption spectroscopy (CU 2-D-MAX-DOAS) instrument measures nitrogen dioxide (NO2), formaldehyde (HCHO), glyoxal (CHOCHO), oxygen dimer (O2–O2, or O4), and water vapor (H2O); nitrous acid (HONO), bromine monoxide (BrO), and iodine monoxide (IO) are among other gases that can in principle be measured. Information about aerosols is derived through coupling with a radiative transfer model (RTM). The 2-D telescope has three modes of operation: mode 1 measures solar scattered photons from any pair of elevation angle (−20° < EA < +90° or zenith; zero is to the horizon) and azimuth angle (−180° < AA < +180°; zero being north); mode 2 measures any set of azimuth angles (AAs) at constant elevation angle (EA) (almucantar scans); and mode 3 tracks the direct solar beam via a separate view port. Vertical profiles of trace gases are measured and used to estimate mixing layer height (MLH). Horizontal distributions are then derived using MLH and parameterization of RTM (Sinreich et al., 2013). NO2 is evaluated at different wavelengths (350, 450, and 560 nm), exploiting the fact that the effective path length varies systematically with wavelength. The area probed is constrained by O4 observations at nearby wavelengths and has a diurnal mean effective radius of 7.0 to 25 km around the instrument location; i.e., up to 1960 km2 can be sampled with high time resolution. The instrument was deployed as part of the Multi-Axis DOAS Comparison campaign for Aerosols and Trace gases (MAD-CAT) in Mainz, Germany, from 7 June to 6 July 2013. We present first measurements (modes 1 and 2 only) and describe a four-step retrieval to derive (a) boundary layer vertical profiles and MLH of NO2; (b) near-surface horizontal distributions of NO2; (c) range-resolved NO2 horizontal distribution measurements using an "onion-peeling" approach; and (d) the ratios HCHO to NO2 (RFN), CHOCHO to NO2 (RGN), and CHOCHO to HCHO (RGF) at 14 pre-set azimuth angles distributed over a 360° view. Three-dimensional distribution measurements with 2-D-MAX-DOAS provide an innovative, regional perspective of trace gases as well as their spatial and temporal concentration gradients, and they maximize information to compare near-surface observations with atmospheric models and satellites.

"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.
"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.

A numerical investigation to mitigate the effects of shock in finite 1:1 dimer chains is performed. Dimer chains consist of alternating light and heavy masses. Changing the mass ratio has provided interesting results in previous research. In particular, in the case of Hertzian contacts with zero-preload, certain mass ratios have revealed minimal levels of transmitted force. This paper examines this phenomena from the perspective of utilizing it in practical isolation systems. The zero-preload Hertzian contact case is contrasted with chains connected by linear or cubic springs. Through numerical simulations, tradeoffs are examined between displacement and transmitted force.