Formation of an adduct by clenbuterol, a beta-adrenoceptor agonist drug, and serum albumin in human saliva at the acidic pH of the stomach: evidence for an aryl radical-based process

Clenbuterol (CLB) is an antiasthmatic drug used also illegally as a lean muscle mass enhancer in both humans and animals. CLB and amine-related drugs in general are nitrosatable, thus raising concerns regarding possible genotoxic/carcinogenic activity. Oral administration of CLB raises the issue of its possible transformation by salivary nitrite at the acidic pH of gastric juice. In acidic human saliva CLB was rapidly transformed to the CLB arenediazonium ion. This suggests a reaction of CLB with salivary nitrite, as confirmed in aerobic HNO(2) solution by a drastic decrease in nitric oxide, nitrite, and nitrate. In human saliva, both glutathione and ascorbic acid were able to inhibit CLB arenediazonium formation and to react with preformed CLB arenediazonium. The effect of ascorbic acid is particularly pertinent because this vitamin is actively concentrated within the gastric juice. EPR spin trapping experiments showed that preformed CLB arenediazonium ion was reduced to the aryl radical by ascorbic acid, glutathione, and serum albumin, the major protein of saliva. As demonstrated by anti-CLB antibodies and MS, the CLB-albumin interaction leads to the formation of a covalent drug-protein adduct, with a preference for Tyr-rich regions. This study highlights the possible hazards associated with the use/abuse of this drug.     

The complex interplay of iron metabolism,reactive oxygen species,and reactive nitrogen species: Insights into the potential of various iron therapies to induce oxidative and nitrosative stress

Production of minute concentrations of superoxide (O₂⁻) and nitrogen monoxide (nitric oxide, NO) plays important roles in several aspects of cellular signaling and metabolic regulation. However, in an inflammatory environment, the concentrations of these radicals can drastically increase and the antioxidant defenses may become overwhelmed. Thus, biological damage may occur owing to redox imbalance—a condition called oxidative and/or nitrosative stress. A complex interplay exists between iron metabolism, O₂⁻, hydrogen peroxide (H₂O₂), and NO. Iron is involved in both the formation and the scavenging of these species. Iron deficiency (anemia) (ID(A)) is associated with oxidative stress, but its role in the induction of nitrosative stress is largely unclear. Moreover, oral as well as intravenous (iv) iron preparations used for the treatment of ID(A) may also induce oxidative and/or nitrosative stress. Oral administration of ferrous salts may lead to high transferrin saturation levels and, thus, formation of non-transferrin-bound iron, a potentially toxic form of iron with a propensity to induce oxidative stress. One of the factors that determine the likelihood of oxidative and nitrosative stress induced upon administration of an iv iron complex is the amount of labile (or weakly-bound) iron present in the complex. Stable dextran-based iron complexes used for iv therapy, although they contain only negligible amounts of labile iron, can induce oxidative and/or nitrosative stress through so far unknown mechanisms. In this review, after summarizing the main features of iron metabolism and its complex interplay with O₂⁻, H₂O₂, NO, and other more reactive compounds derived from these species, the potential of various iron therapies to induce oxidative and nitrosative stress is discussed and possible underlying mechanisms are proposed. Understanding the mechanisms, by which various iron formulations may induce oxidative and nitrosative stress, will help us develop better tolerated and more efficient therapies for various dysfunctions of iron metabolism.     

Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation

The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO(2)(-). The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions.     

Detection of a genetic variant, lysine→glutamic acid at position 372 of human serum albumin, by capillary electrophoresis and structural identification

A genetic variant of human serum albumin (alloalbumin) is detected by capillary electrophoresis (CE). Two albumin peaks, which were in the ratio of approximately one, were clearly separated. One of the peaks had the same migration time as normal albumin (Alb A) and the other (Alb X) had a longer migration time. SDS-polyacrylamide gel electrophoresis of CNBr fragments (CB) of Alb X indicated that the amino acid substitution was localized in the CB5 fragment (residue 330–446) of the molecule, because of anomalous migration of CB5 in the gel. The CE mapping of the tryptic peptides from the variant CB5 revealed clearly the existence of a new peptide, and the lack of two normal peptides. The sequence analysis of the variant peptide collected by CE micropreparation showed that the N-terminus of the variant peptide corresponded to that of T49 in Alb A. The substitution site, lysine→glutamic acid at the position 372, was revealed by sequence determination of the variant peptide purified by reversed-phase HPLC.     

Methylglyoxal-bovine serum albumin stimulates tumor necrosis factor alpha secretion in RAW 264.7 cells through activation of mitogen-activating protein kinase,nuclear factor kappaB and intracellular reactive oxygen species formation

Accumulating evidence suggests that the pathophysiology of diabetes is analogous to chronic inflammatory states. Circulating levels of inflammatory cytokines such as IL-6 and tumor necrosis factor alpha (TNFalpha) are increased in both type 1 and type 2 diabetes. TNFalpha plays an important role in the pathogenesis of insulin resistance in type 2 diabetes. However, the reason for this increase remains unclear. Levels of the dicarbonyl methylglyoxal (MGO) are elevated in diabetic plasma and MGO-modified bovine serum albumin (MGO-BSA) can trigger cellular uptake of TNF. Therefore we tested the hypothesis that MGO-modified proteins may cause TNFalpha secretion in macrophage-like RAW 264.7 cells. Treatment of cells with MGO-BSA induced TNFalpha release in a dose-dependent manner. MGO-modified ribonuclease A and chicken egg ovalbumin had similar effects. Cotreatment of cells with antioxidant reagent N-acetylcysteine (NAC) inhibited MGO-BSA-induced TNFalpha secretion. MGO-BSA stimulated the simultaneous activation of p44/42 and p38 mitogen-activated protein kinase. PD98059, a selective MEK inhibitor, inhibited MGO-BSA-induced TNFalpha release as well as ERK phosphorylation. Pretreatment of cells with NAC also resulted in inhibition of MGO-BSA-induced ERK phosphorylation. MGO-BSA induced dose-dependent NFkappaB activation as shown by electrophoresis mobility shift assay. The MGO-BSA-induced NFkappaB activation was prevented in the presence of PD98059, NAC, and parthenolide, a selective inhibitor of NFkappaB. Furthermore, the NFkappaB inhibitor parthenolide suppressed MGO-BSA-induced TNFalpha secretion. Confocal microscopy using dichlorofluorescein to demonstrate intracellular reactive oxygen species (ROS) showed that MGO-BSA produced more ROS compared with native BSA. MGO-BSA could also stimulate protein kinase C (PKC) translocation to the cell membrane, considered a key signaling pathway in diabetes. However, there was no evidence that PKC was involved in TNFalpha release based on inhibition by calphostin C and staurosporine. Our findings suggest that the presence of chronically elevated levels of MGO-modified bovine serum albumin may contribute to elevated levels of TNFalpha in diabetes.     

Development of PEGylated serum albumin with multiple reduced thiols as a long-circulating scavenger of reactive oxygen species for the treatment of fulminant hepatic failure in mice

Reactive oxygen species (ROS) are involved in the pathophysiology of fulminant hepatic failure. Therefore, we developed polyethylene glycol-conjugated bovine serum albumin with multiple reduced thiols (PEG-BSA-SH) for the treatment of fulminant hepatic failure. As a long-circulating ROS scavenger, PEG-BSA-SH effectively scavenged highly reactive oxygen species and hydrogen peroxide in buffer solution. PEG-BSA-SH showed a long circulation time in the plasma after intravenous injection into mice. Fulminant hepatic failure was induced by intraperitoneal injection of lipopolysaccharide and d-galactosamine (LPS/d-GalN) into mice. The LPS/d-GalN-induced elevation of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was significantly inhibited by a bolus intravenous injection of PEG-BSA-SH. Furthermore, the changes in hepatic lipid peroxide and hepatic blood flow were effectively suppressed by PEG-BSA-SH. In contrast, l-cysteine, glutathione, and dithiothreitol, three traditional reduced thiols, had no statistically significant effects on the serum levels of ALT or AST. These findings indicate that PEG-BSA-SH is a promising ROS scavenger and useful in the treatment of fulminant hepatic failure.     

Induction of bystander effects by UVA,UVB, and UVC radiation in human fibroblasts and the implication of reactive oxygen species

Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far have been reported mainly after UVA and UVB radiation. The studies described here were aimed at comparing the responses of human dermal fibroblasts exposed directly to UV (A, B, or C wavelength range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation system. Cell survival and apoptosis were used as a measure of radiation effects. Additionally, induction of senescence in UV-exposed and bystander cells was evaluated. Reactive oxygen species (ROS), superoxide radical anions, and nitric oxide inside the cells and secretion of interleukins 6 and 8 (IL-6 and IL-8) into the medium were assayed and evaluated as potential mediators of bystander effects. All three regions of ultraviolet radiation induced bystander effects in unexposed cells, as shown by a diminution of survival and an increase in apoptosis, but the pattern of response to direct exposure and the bystander effects differed depending on the UV spectrum. Although UVA and UVB were more effective than UVC in generation of apoptosis in bystander cells, UVC induced senescence both in irradiated cells and in neighbors. The level of cellular ROS increased significantly shortly after UVA and UVB exposure, suggesting that the bystander effects may be mediated by ROS generated in cells by UV radiation. Interestingly, UVC was more effective at generation of ROS in bystanders than in directly exposed cells and induced a high yield of superoxide in exposed and bystander cells, which, however, was only weakly associated with impairment of mitochondrial membrane potential. Increasing concentration of IL-6 but not IL-8 after exposure to each of the three bands of UV points to its role as a mediator in the bystander effect. Nitric oxide appeared to play a minor role as a mediator of bystander effects in our experiments. The results demonstrating an increase in intracellular oxidation, not only in directly UV-exposed but also in neighboring cells, and generation of proinflammatory cytokines, processes entailing cell damage (decreased viability, apoptosis, senescence), suggest that all bands of UV radiation carry a potential hazard for human health, not only due to direct mechanisms, but also due to bystander effects.     

The relationship between uric acid and its oxidative product allantoin: a potential indicator for the evaluation of oxidative stress in birds

Uric acid is the main nitrogenous waste product in birds but it is also known to be a potent antioxidant. Hominoid primates and birds lack the enzyme urate oxidase, which oxidizes uric acid to allantoin. Consequently, the presence of allantoin in their plasma results from non-enzymatic oxidation. In humans, the allantoin to uric acid ratio in plasma increases during oxidative stress, thus this ratio has been suggested to be an in vivo marker for oxidative stress in humans. We measured the concentrations of uric acid and allantoin in the plasma and ureteral urine of white-crowned sparrows (Zonotrichia leucophrys gambelii) at rest, immediately after 30 min of exercise in a hop/hover wheel, and after 1 h of recovery. The plasma allantoin concentration and the allantoin to uric acid ratio did not increase during exercise but we found a positive relationship between the concentrations of uric acid and allantoin in the plasma and in the ureteral urine in the three activity phases. In the plasma, the slope of the regression describing the above positive relationships was significantly higher immediately after activity. We suggest that the slope indicates the rate of uric acid oxidation and that during activity this rate increases as a result of higher production of free radicals. The present study demonstrates that allantoin is present in the plasma and in the ureteral urine of white-crowned sparrows and therefore might be useful as an indicator of oxidative stress in birds.     

Novel insights into the pleiotropic effects of human serum albumin in health and disease

Background

Human serum albumin is the principal protein in human serum. It participates in regulation of plasma oncotic pressure and transports endogenous and exogenous ligands such as thyroxine, free fatty acids, bilirubin, and various drugs. Therefore, studying its ligand binding mechanism is important in understanding many functions of the protein.

Scope of review

This review discusses the pleiotropic biochemical effects and their relevance to physiologic functions of albumin.

Major conclusions

Although HSA is traditionally recognized for its ligand transport and oncotic effects in human circulation, our studies have revealed its participation in several other important physiological functions. In some instances, it may function as a catalyst. Pleiotropic properties of HSA have been exploited by development of recombinant HSA and its mutants, and the use of these recombinant proteins in studies with various biochemical and biophysical techniques. These studies allowed us to obtain new insights on the diverse roles of HSA in human physiology. The following aspects of HSA were discussed in this review: 1) HSA and its mutants' role in thyroxine transport, 2) structural details of the ligand binding functions of HSA to ligands such as warfarin, digoxin, halothane anesthetics, nitric oxide, bilirubin, free fatty acids, etc, and 3) the formation of modified albumin during myocardial ischemia, its diagnostic significance, and HSA's role in cardiovascular disease.

General significance

The appreciation and understanding of structural details and new physiological roles has provided a renewed interest in HSA research. Specific structural information gained on various mechanisms of HSA–ligand interaction can be used to develop a model to better understand protein–drug interactions, aid in the development of new drugs with improved pharmacokinetic effects, and ultimately be used to improve the quality of healthcare. This article is part of a Special Issue entitled Serum Albumin.     

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (K_a), ΔH, ΔS and ΔG values to be 15.5×10⁴M^?1, ?116.2±18.1 Kcal mol^?1, ?366 cal mol^?1K^?1 and ?7.13 Kcal mol^?1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.     

DNA damage induced by catecholestrogens in the presence of copper (II): generation of reactive oxygen species and enhancement by NADH

Certain estrogen metabolites are involved in carcinogenesis and the development of resistance to methotrexate (MTX). In this study, we determined whether these well-established biological effects correlate with the relative efficiency of several estrogen metabolites to induce DNA strand breaks in the presence of copper, and investigated the potential enhancing effect of reduced nicotinamide adenine dinucleotide (NADH). DNA strand breaks induced by estradiol metabolites were measured by the conversion of supercoiled phage phiX-174 RF1 DNA to open circular and linear forms. The most active catecholestrogens were the 4-hydroxy derivatives, which produced about 2.5 times more DNA double strand breaks than the 2-hydroxy derivatives, while estradiol and 16alpha-hydroxyestrone were inactive. In addition, our results show that 4-hydroxyestradiol (4-OHE2) at physiological concentrations was capable of exhibiting DNA cleaving activity. The formation of these catecholestrogen-induced DNA strand breaks was associated with the utilization of oxygen and the generation of H2O2, because catalase inhibited the DNA cleaving activity of 4-OHE2. Interestingly, we also observed that NADH enhanced the induction of DNA strands breaks by 4-OHE2/Cu(II), probably by perpetuating the redox cycle between the quinone and the semiquinone forms of the catecholestrogen. In conclusion, this study demonstrated that the relative efficiency of 2-, and 4-hydroxyestrogen in carcinogenesis and for the enhancement of MTX resistance correlates with their relative capability to induce DNA strand breaks. In order to inhibit these estrogen-mediated biological effects, it may be important to develop different strategies to block the production of reactive oxygen species by the catecholestrogen-redox cycle.     

Interactions of urocanic acid with bovine serum albumin and the influence of pH on binding affinities: a docking simulation study

In the present study, the interactions of urocanic acid (UA) with bovine serum albumins (BSA) at pH 5.0 and 7.4 were investigated by means of docking simulations. The binding modes of trans- and cis-UA to BSA at pH 5.0 and 7.4 were analysed. In addition, the theoretically predicted binding abilities of zwitterion and anion of UA with BSA are in good agreement with the experimental results. Through comparison with the binding patterns, we revealed that the stronger interactions of UA anion with BSA relative to the zwitterion primarily result from: (1) the increased number of hydrogen bonds between UA anion and BSA; (2) the attractive electrostatic interaction between the deprotoned carboxyl group in UA anion and Arg433 in comparison with the repulsion between the imidazole moiety in zwitterion and the same residue in BSA. This provides a rational explanation for the experimental finding that the binding of UA to BSA at pH 7.4 is much stronger than at pH 5.0.     

N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-l-tryptophan

Background

Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products.

Methods

Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4 weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration.

Results

N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present.

Conclusion

The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations.

General significance

N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.     

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography–mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18–23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential.     

Evidence for the involvement of nitric oxide and reactive oxygen species in osmotic stress tolerance of wheat seedlings: Inverse correlation between leaf abscisic acid accumulation and leaf water loss

Nitric oxide (NO) and reactive oxygen species (ROS) play important roles in both abscisic acid (ABA) signaling and stress-induced ABA accumulation. However, little is known about their physiological roles in the whole plant. In this study, the effects of NO and ROS on leaf water control and the roles of ABA were determined using wheat (Triticum aestivum L.) seedlings. As compared with the control, osmotic stress reduced leaf water loss (LWL) while it increased leaf ABA content. The effects of osmotic stress on LWL and ABA contents were partially reversed by NO scavengers or NO synthase (NOS) inhibitors. Furthermore, sodium nitroprusside (SNP) at concentrations between 0.01 and 10 mM all reduced LWL efficiently and induced ABA accumulation in a dose-dependent manner. When ABA synthesis was inhibited by fluridone or actidione, the effects of SNP on LWL were partially reversed. These results suggest that NO is involved in leaf water maintenance of wheat seedlings under osmotic stress, and one of the possible mechanisms is by stimulating ABA synthesis. The ROS scavengers used in our experiments had no effects on either LWL or ABA accumulation induced by osmotic stress. However, all ROS induced LWL reduction and ABA accumulation significantly. Hydrogen peroxide had the same effects as SNP on LWL and induced ABA accumulation in a dose-dependent manner but had a maximal effect at 1 mM. Fluridone reversed the effects of H₂O₂ on both LWL reduction and ABA accumulation, while actidione had no effect. These results suggest that ROS are also involved in leaf water maintenance of wheat seedlings by stimulating ABA biosynthesis, but with a different mechanism to that of NO. The ABA-independent mechanism in NO/ROS regulation of leaf water balance is discussed, in relation to our results.     

Mitochondrial generation of reactive oxygen species is enhanced at the Q<Subscript>o</Subscript> site of the complex III in the myocardium of <Emphasis Type="Italic">Trypanosoma cruzi-</Emphasis>infected mice: beneficial effects of an antioxidant

In this study, we have characterized the cellular source and mechanism for the enhanced generation of reactive oxygen species (ROS) in the myocardium during Trypanosoma cruzi infection. Cardiac mitochondria of infected mice, as compared to normal controls, exhibited 63.3% and 30.8% increase in ROS-specific fluorescence of dihydroethidium (detects O₂ ^•−) and amplex red (detects H₂O₂), respectively. This increase in ROS level in cardiac mitochondria of infected mice was associated with a 59% and 114% increase in the rate of glutamate/malate- (complex I substrates) and succinate- (complex II substrate) supported ROS release, respectively, and up to a 74.9% increase in the rate of electron leakage from the respiratory chain when compared to normal controls. Inhibition studies with normal cardiac mitochondria showed that rotenone induced ROS generation at the Q_Nf-ubisemiquinone site in complex I. In complex III, myxothiazol induced ROS generation from a site located at the Q_o center that was different from the Q_i center of O₂ ^•− generation by antimycin. In cardiac mitochondria of infected mice, the rate of electron leakage at complex I during forward (complex I-to-complex III) and reverse (complex II-to-complex I) electron flow was not enhanced, and complex I was not the main site of increased ROS production in infected myocardium. Instead, defects of complex III proximal to the Q_o site resulted in enhanced electron leakage and ROS formation in cardiac mitochondria of infected mice. Treatment of infected mice with phenyl-α-tert-butyl-nitrone (PBN) improved the respiratory chain function, and, subsequently, decreased the extent of electron leakage and ROS release. In conclusion, we show that impairment of the Q_o site of complex III resulted in increased electron leakage and O₂ ^•− formation in infected myocardium, and was controlled by PBN.     

Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: A combined spectroscopic and docking simulation approach

A comparative biophysical study on the individual conformational adaptation embraced by two homologous serum albumins (SA) (bovine and human) towards a potential anticancer bioorganic compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)- dione (CBIQD) is apparent from the discrimination in binding behavior and the ensuing consequences accomplished by combined in vitro optical spectroscopy, in silico molecular docking and molecular dynamics (MD) simulation. The Sudlow site I of HSA although anion receptive, harbors neutral CBIQD in Sudlow site I (subdomain IIA, close to Trp) of HSA, while in BSA its prefers to snugly fit into Sudlow site II (subdomain IIIA, close to Tyr). Based on discernable diminution of HSA mean fluorescence lifetime as a function of biluminophore concentration, facile occurrence of fluorescence resonance energy transfer (FRET) is substantiated as the probable quenching mechanism accompanied by structural deformations in the protein ensemble. CBIQD establishes itself within HSA close to Trp214, and consequently reduces the micropolarity of the cybotactic environment that is predominantly constituted by hydrophobic amino acid residues. The stronger association of CBIQD with HSA encourages an allosteric modulation leading to slight deformation in its secondary structure whereas for BSA the association is comparatively weaker. Sudlow site I of HSA is capable to embrace a favorable conformation like malleable gold to provide room for incoming CBIQD, whereas for BSA it behaves more like rigid cast-iron which does not admit any change thus forcing CBIQD to occupy an altogether different binding location i.e. the Sudlow site II. The anticancer CBIQD is found to be stable within the HSA scaffold as vindicated by root mean square deviation (RMSD) and root mean square fluctuation (RMSF) obtained by MD simulation. A competitively inhibited esterase-like activity of HSA upon CBIQD binding to Lys199 and Arg257 residues, plausibly envisions that similar naphthalimide based prodrugs, bearing ester functionality, can be particularly activated by Sudlow site I of HSA. The consolidated spectroscopic research described herein may encourage design of naphthalimide based pro-drugs for effective in vivo biodistribution using HSA-based drug delivery systems.