Synthesis, crystal structure, and magnetic properties of a new tetranuclear Cu(II) Schiff base compound

A new tetranuclear Cu(II) compound [Cu₄(HL)₂(L)₂(ClO₄)₂] (1) was synthesized from the reaction of Cu(ClO₄)₂ · 6H₂O with Schiff base ligand (H₂L) condensed from ethanolamine with 2-hydroxyacetophenone. X-ray diffraction studies revealed that 1 is formed from the self-assembly of two dinuclear units [Cu₂(HL)(L)(ClO₄)] through the doubly phenoxo bridging. The variable temperature magnetic susceptibility measurements were performed between 300 K and 2 K and show χ_MT value for 1 at 300 K is 1.395 cm³ mol⁻¹ K and fall to 0.0459 cm³ mol⁻¹ K at 2 K. These values are smaller than that expected for tetranuclear copper (II) units, indicating antiferromagnetic coupling present in the compound. This result is also confirmed from the DFT calculations.     

Polyphenolic disaccharides endow proteins with unusual resistance to aggregation

Protein aggregation is a common problem during the purification and formulation of therapeutic proteins. Here we report that polyphenolic disaccharides are unusually effective at preventing protein aggregation. We find that two polyphenolic glycosides-naringin and rutin-endow diverse proteins with the ability to unfold without aggregating when heated, as well as the ability to refold without aggregating when cooled at low glycoside concentrations (<5 mM). This extreme solubilizing activity is a synergistic combination of the glycone and aglycone moieties, as combinations of polyphenols and sugars fail to suppress aggregation. Moreover, the activity of polyphenolic disaccharides is remarkably specific since their monosaccharide counterparts (as well as other common excipients such as arginine, trehalose, and cyclodextrin) fail to prevent aggregation at similar concentrations (<25 mM). We expect that polyphenolic disaccharides will be valuable additives for enhancing the solubility of proteins in applications plagued by protein aggregation.     

Distinct synergistic action of piperacillin and methylglyoxal against Pseudomonas aeruginosa

The dicarbonyl compound methylglyoxal is a natural constituent of Manuka honey produced from Manuka flowers in New Zealand. It is known to possess both anticancer and antibacterial activity. Such observations prompted to investigate the ability of methylglyoxal as a potent drug against multidrug resistant Pseudomonas aeruginosa. A total of 12 test P. aeruginosa strains isolated from various hospitals were tested for their resistances against many antibiotics, most of which are applied in the treatment of P. aeruginosa infections. Results revealed that the strains were resistant to many drugs at high levels, only piperacillin, carbenicillin, amikacin and ciprofloxacin showed resistances at comparatively lower levels. Following multiple experimentations it was observed that methylglyoxal was also antimicrobic against all the strains at comparable levels. Distinct and statistically significant synergism was observed between methylglyoxal and piperacillin by disc diffusion tests when compared with their individual effects. The fractional inhibitory concentration index of this combination evaluated by checkerboard analysis, was 0.5, which confirmed synergism between the pair. Synergism was also noted when methylglyoxal was combined with carbenicillin and amikacin.     

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO(2)-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO(2)-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.     

Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions

High-affinity antibodies are critical for numerous diagnostic and therapeutic applications, yet their utility is limited by their variable propensity to aggregate either at low concentrations for antibody fragments or high concentrations for full-length antibodies. Therefore, determining the sequence and structural features that differentiate aggregation-resistant antibodies from aggregation-prone ones is critical to improving their activity. We have investigated the molecular origins of antibody aggregation for human V(H) domain antibodies that differ only in the sequence of the loops containing their complementarity determining regions (CDRs), yet such antibodies possess dramatically different aggregation propensities in a manner not correlated with their conformational stabilities. We find the propensity of these antibodies to aggregate after being transiently unfolded is not a distributed property of the CDR loops, but can be localized to aggregation hotspots within and near the first CDR (CDR1). Moreover, we have identified a triad of charged mutations within CDR1 and a single charged mutation adjacent to CDR1 that endow the poorly soluble variant with the desirable biophysical properties of the aggregation-resistant antibody. Importantly, we find that several other charged mutations in CDR1, non-CDR loops and the antibody scaffold are incapable of preventing aggregation. We expect that our identification of aggregation hotspots that govern antibody aggregation within and proximal to CDR loops will guide the design and selection of antibodies that not only possess high affinity and conformational stability, but also extreme resistance to aggregation.     

Leishmania donovani Infection Enhances Lateral Mobility of Macrophage Membrane Protein Which Is Reversed by Liposomal Cholesterol

BackgroundThe protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.Conclusions/SignificancesTo our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.     

Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana

Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.     

Function of S-nitrosoglutathione reductase (GSNOR) in plant development and under biotic/abiotic stress

During the last decade, it was established that the class III alcohol dehydrogenase (ADH3) enzyme, also known as glutathione-dependent formaldehyde dehydrogenase (FALDH; EC 1.2.1.1), catalyzes the NADH-dependent reduction of S-nitrosoglutathione (GSNO) and therefore was also designated as GSNO reductase. This finding has opened new aspects in the metabolism of nitric oxide (NO) and NO-derived molecules where GSNO is a key component. In this article, current knowledge of the involvement and potential function of this enzyme during plant development and under biotic/abiotic stress is briefly reviewed.Key words: nitric oxide, nitrosative stress, S-nitrosoglutathione reductase