A Healthy and Ecologically Balanced Environment: An Argument for a Third Generation Right

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr(4)-Ile(5) bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants

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SARS-CoV-2-specific immune responses and clinical outcomes after COVID-19 vaccination in patients with immune-suppressive disease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune responses and infection outcomes were evaluated in 2,686 patients with varying immune-suppressive disease states after administration of two Coronavirus Disease 2019 (COVID-19) vaccines. Overall, 255 of 2,204 (12%) patients failed to develop anti-spike antibodies, with an additional 600 of 2,204 (27%) patients generating low levels (<380 AU ml−1). Vaccine failure rates were highest in ANCA-associated vasculitis on rituximab (21/29, 72%), hemodialysis on immunosuppressive therapy (6/30, 20%) and solid organ transplant recipients (20/81, 25% and 141/458, 31%). SARS-CoV-2-specific T cell responses were detected in 513 of 580 (88%) patients, with lower T cell magnitude or proportion in hemodialysis, allogeneic hematopoietic stem cell transplantation and liver transplant recipients (versus healthy controls). Humoral responses against Omicron (BA.1) were reduced, although cross-reactive T cell responses were sustained in all participants for whom these data were available. BNT162b2 was associated with higher antibody but lower cellular responses compared to ChAdOx1 nCoV-19 vaccination. We report 474 SARS-CoV-2 infection episodes, including 48 individuals with hospitalization or death from COVID-19. Decreased magnitude of both the serological and the T cell response was associated with severe COVID-19. Overall, we identified clinical phenotypes that may benefit from targeted COVID-19 therapeutic strategies

Co-Design Architecture and Implementation for Point-Based Rendering on FPGAs

Current graphic cards include advanced graphic processing units to accelerate the rendering of 3D objects with millions of polygons. As object models grow in complexity, the rendering approach based on points as primitives is regarded superior in terms of scalability and efficiency. Next generation graphic cards could contain reconfigurable fabrics, similar to those implemented in current FPGAs, to offer two advantages: a) fast rendering units and b) new mechanisms for custom, run-time exchangeable accelerators. In this paper, we propose a hardware point-rendering architecture tailored specifically for reconfigurable systems. The presented implementation on a real FPGA-based platform demonstrates on the one hand the effectiveness of the approach and on the other hand it provides valuable insights into possible future improvements for this problem scenario

Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr(4)-Ile(5) bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes

The effect of mutations in Arg143 and Lys192 mutants on the processing of Ang I.

<p>The Arg143 and Lys192 variants ability to process Ang I was analysed by cleaving 10 µg of Ang I in a 20 µl reaction volume. Ang I (Asp₁-Arg₂-Val₃-Tyr₄-Ile₅-His₆-Pro₇-Phe₈-His₉-Leu₁₀, MW∼1297) is converted to Ang II (MW∼1046) by cleavage after the Phe₈ bond. As reference samples we also used the wt enzyme and the double mutant (Arg143+Lys192). Approximately the same amount of protease, as determined by cleavage of the chromogenic substrate LLVY-pNA, was used in each reaction (Right panel). Two µl samples were removed at different time points and analysed by mass spectrometry. The obtained results were compared in order to estimate the generated products aswell as a semi-quantitative measurement of the cleavage rate and its dependence on residues Arg143 and Lys192 and the double mutant. These experiments were independently performed three times with similar results.</p

Structures of the five different chymase inhibitors used in this study.

<p>Five different patented inhibitors of the HC originating from 5 different companies were synthesized. The inhibitors were subsequently used to determine the dependence of the basic amino acids Arg143 and Lys192 for the specificity in the interaction between the enzyme and the inhibitor. Compound A (TY51184) originates from Tao Eiyo (patent no WO 2002 022595), compound B from Teijin (patent no WO 2007 068621), compound C from Johnson & Johnson (patent no WO 2005 073214), compound D from Roche (patent no WO 2000 003997) and compound E from Boehringer Ingelheim (patent no WO 2009 023655).</p