Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO ؊ ) on the formation of N -(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO ؊ , CML formation was detected by both enzymelinked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO ؊ concentrations. CML was also formed when glucose, preincubated with ONOO ؊ , was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for ␣-oxoaldehydes. For identifying the aldehydes that contributed to ONOO ؊ -induced CML formation, glucose was incubated with ONOO ؊ in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO ؊ can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive ␣-oxoaldehydes from glucose.
We investigated the numbers of planktonic and biofilm cells and the expression levels of genes encoding efflux pumps and biofilm-related proteins in 10 clinical isolates of multi-drug resistant Acinetobacter baumannii (MDRA) as well as in its standard strain ATCC 19606 in the presence of colistin (CST), polymyxin B (PMB), minomycin (MIN), and tigecycline (TGC) at their respective sub-MICs. The number of planktonic and biofilm cells of ATCC 19606 decreased in the presence of all aforementioned antibiotics in a dose-dependent manner. Cell number also decreased in two representative MDRA strains, R2 and R3, in the presence of MIN and TGC in a dose-dependent manner. In contrast, the number of biofilm cells in these two strains increased in the presence of CST, while they increased significantly in the presence of PMB in R2 only. Pearson correlation analysis revealed that the number of biofilm cells was positively and significantly correlated with the mRNA levels of genes encoding efflux pumps (adeB and adeG) and autoinducer synthase (abaI) in strain R2 and adeB, adeG, adeJ, poly-acetyl-glucosamine-porin (pgaA), and abaI in strain R3 in the presence of CST. It was positively and significantly correlated with the mRNA levels of genes encoding adeB in strain R2 and an outer membrane protein A (ompA) and biofilm-associated protein (bap) in strain R3 in the presence of PMB. These results provide valuable insights into the biofilm formation potency of clinical isolates of MDRA that depends on efflux pumps and biofilm-related genes and its regulation by antibiotics.
We investigated the intracellular survival of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates in macrophages, after phagocytosis, to determine their virulence characteristics. After ATCC 19606 and 5 clinical isolates of MDRAB were phagocytosed by mouse and human macrophages, the bacterial count of MDRAB strains, R4 and R5, increased in the mouse macrophages, 24 hours after phagocytosis. Bacterial count of the strains, R1 and R2, was almost equal 4 and 24 hours after phagocytosis. Intracellular reactive oxygen species was detected in the macrophages after phagocytosis of these bacteria. Further, the strains R1, R2, R4, and R5 showed higher catalase activity than ATCC 19606. Additionally, strains R1, R4, and R5 grew more efficiently than ATCC 19606 in the presence of H2O2, whereas growth of strains R2 and R3 was marginally more than that of ATCC 19606 in the presence of H2O2. The MDRAB clinical isolates altered the expression of TNF-α, IL-1β, IL-6, and MIP-2 mRNA induced in J774A.1 cells, 24 hours after phagocytosis. These results provide insights into the renewed virulence characteristics of MDRAB clinical isolates. Finally, tigecycline killed MDRAB phagocytosed by the macrophages more effectively than colistin, although colistin and tigecycline are both considered effective antibiotics for the treatment of MDRAB.
To develop potent covalent inhibitors, the noncovalent interactions around the transition state to form covalent bonding should be optimized because the potency of the inhibitor can be depending on the energy of the transition state. Here, we report an efficient analysis of the noncovalent binding mode of a potent covalent proteasome inhibitor 3a around the transition state by a combined use of the chemical approach, i.e., the cyclopropylic strain-based conformational restriction, and the computational docking approach. Furthermore, we calculated the binding energy of a series of salinosporamide derivatives in the predicted noncovalent complex around the transition state with the simulation model of proteasome constructed in this study, which was well correlated to their pIC50. Thus, the proposed docking methods to predict the noncovalent binding mode around the transition state of covalent inhibitors will be helpful toward the development of covalent inhibitors.
These results provide insights into the renewed virulence characteristics of A. baumannii clinical isolates that depend on cell adherence capacity and the expression level of omp mRNAs.
A series of chiral 2,3- and 3,4-methanoamino acid equivalents of stereochemical diversity were designed and synthesized from our chiral cyclopropane units, using a diastereoselective Grignard addition with ( R)- or ( S)- t-butanesulfinyl imines as the key step. These equivalents were converted into the proteasome inhibitor belactosin A and its cis-cyclopropane stereoisomer. The unnatural cis-isomer was shown to be more than twice as potent as belactosin A as a proteasome inhibitor.
The pathological significance of advanced glycation end product (AGE)-modified proteins deposited in several lesions is generally accounted for by their cellular interaction via the AGE receptors and subsequent acceleration of the inflammatory process. In this study, we focused on two AGE receptors-specifically, the role of SR-A in pathogenesis of diabetic nephropathy and the role of CD36 in AGE-induced downregulation of leptin by adipocytes. In terms of SR-A, diabetic wild-type mice exhibited increased urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion, whereas SR-A-knockout mice showed reduced glomerular size and mesangial matrix area. In these diabetic SR-A-knockout mice, the number of macrophages that infiltrated into glomeruli was remarkably reduced (P < 0.05), suggesting that SR-A-dependent glomerular migration of macrophages plays an important role in the pathogenesis of diabetic nephropathy. In terms of CD36, incubation of glycolaldehyde-modified bovine serum albumin (GA-BSA) with 3T3-L1 adipocytes reduced leptin secretion by these cells. The binding of GA-BSA to these cells and subsequent endocytic degradation were effectively inhibited by a neutralizing anti-CD36 antibody. AGE-induced downregulation of leptin was protected by N-acetyl-cysteine, an antioxidant. These results indicate that the interaction of AGE ligands with 3T3-L1 adipocytes via CD36 induces oxidative stress and leads to inhibition of leptin expression by these cells, suggesting a potential link of this phenomenon to exacerbation of the insulin sensitivity in metabolic syndrome.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.