Non-enzymatic Protein Research Articles

Proteostasis in the Male and Female Germline: A New Outlook on the Maintenance of Reproductive Health.

For fully differentiated, long lived cells the maintenance of protein homeostasis (proteostasis) becomes a crucial determinant of cellular function and viability. Neurons are the most well-known example of this phenomenon where the majority of these cells must survive the entire course of life. However, male and female germ cells are also uniquely dependent on the maintenance of proteostasis to achieve successful fertilization. Oocytes, also long-lived cells, are subjected to prolonged periods of arrest and are largely reliant on the translation of stored mRNAs, accumulated during the growth period, to support meiotic maturation and subsequent embryogenesis. Conversely, sperm cells, while relatively ephemeral, are completely reliant on proteostasis due to the absence of both transcription and translation. Despite these remarkable, cell-specific features there has been little focus on understanding protein homeostasis in reproductive cells and how/whether proteostasis is “reset” during embryogenesis. Here, we seek to capture the momentum of this growing field by highlighting novel findings regarding germline proteostasis and how this knowledge can be used to promote reproductive health. In this review we capture proteostasis in the context of both somatic cell and germline aging and discuss the influence of oxidative stress on protein function. In particular, we highlight the contributions of proteostasis changes to oocyte aging and encourage a focus in this area that may complement the extensive analyses of DNA damage and aneuploidy that have long occupied the oocyte aging field. Moreover, we discuss the influence of common non-enzymatic protein modifications on the stability of proteins in the male germline, how these changes affect sperm function, and how they may be prevented to preserve fertility. Through this review we aim to bring to light a new trajectory for our field and highlight the potential to harness the germ cell’s natural proteostasis mechanisms to improve reproductive health. This manuscript will be of interest to those in the fields of proteostasis, aging, male and female gamete reproductive biology, embryogenesis, and life course health.

Discovery of Flazin, an Alkaloid Isolated from Cherry Tomato Juice, As a Novel Non-Enzymatic Protein Glycation Inhibitor via in Vitro and in Silico Studies.

Both overproduced reactive oxygen species/reactive nitrogen species and hyperglycemic conditions accompany a significant increase in protein glycation and nitration that contribute to the initiation and progression of diabetic complications and neuronal disorders. In this study, 19 compounds, including steroidal saponins, alkaloids, cerebroside, phenolic compounds, sterols, and nucleosides, were isolated from cherry tomato (Solanum lycopersicum var. cerasiforme) juice, of which flazin showed good inhibition on monosaccharide-induced non-enzymatic bovine pancreas insulin and bovine serum albumin (BSA) glycation. Molecular dynamics simulations revealed that flazin continuously interacts with Phe1, Val2, Tyr26, and Lys29 insulin residues, which play a key role in insulin glycation/dimerization. In addition, depending upon the flazin dose, this blocked the tyrosine nitration of BSA via scavenging peroxynitrite anions. Taken together, our novel findings suggest that flazin could be a lead compound for the treatment of diabetes and neuronal disorders via the inhibition of non-enzymatic protein glycation and the elimination of peroxynitrite.

The expansin gene NtEXPA5 increases stress tolerance of tobacco hairy roots through an effect on the antioxidant system

BACKGROUND: Expansins are non-enzymatic proteins involved in the softening of cell walls, the mechanism of action of which is associated with the weakening and breaking of hydrogen bonds between xyloglucans and cellulose microfibrils and is aimed at ensuring cell expansion.
 THE AIM of our work was to obtain hairy roots of tobacco with constitutive expression of the NtEXPA5 expansin gene, their morphometric analysis and assessment of the state of their antioxidant system in response to stress factors.
 MATERIALS AND METHODS: The hairy roots were obtained from transgenic tobacco plants expressing the NtEXPA5 gene under the control of the 35S promoter.
 RESULTS: Constitutive expression of the NtEXPA5 gene promoted an increase in the length and dry weight of hairy roots both under normal conditions and under the action of salinity, copper sulfate, cadmium acetate, and mannitol. Both under normal conditions and under the action of stress factors in transgenic hairy roots, an increase in the activity of superoxide dismutase and the total antioxidate activity was recorded.
 CONCLUSION: Expansins exert their positive effect on the productivity and stress tolerance of plants not only through their influence on cell expansion, but also through the effect on the antioxidant system.

Overexpression of an expansin-like gene, GhEXLB2 enhanced drought tolerance in cotton

Expansins are nonenzymatic cell wall proteins that play significant role in plant development as well as stress responses. Hereby, an expansin-like gene, GhEXLB2 was isolated from a cotton (Gossypium hirsutum L.) protoplast with suppression subtractive hybridization to characterize and study its responses against abiotic stresses. GhEXLB2 is the cell-wall localized protein. The expression of GhEXLB2 level was significantly high under polyethylene glycol and salt treatments. GhEXLB2 was further characterized in vitro by cloning and transformation into cotton. Cotton plants overexpressing GhEXLB2 showed enhanced drought tolerance at germination, seedling and flowering stages. After polyethylene glycol (PEG) treatment at germination stage, the length of main root and hypocotyl of overexpressing lines was significantly longer than YZ1 (wild type) and RNAi lines. In addition, H2O2 and malondialdehyde (MDA) contents were lower, while superoxide dismutase (SOD) and peroxidase (POD) activity was detected higher in overexpressing seedlings. On the other hand, higher SOD and POD activity was detected in overexpressing lines than WT plants in soil. In addition, water use efficiency (WUE), soluble sugar, and chlorophyll contents were also significantly greater in overexpressing plants. The present study revealed that GhEXLB2 play crucial role in enhancing drought resistivity in cotton.

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid

ABSTRACT: In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.

FTSC-Labeling Coupled with 2DE-LC-MS/MS Analysis of Complex Protein Mixtures for Identification and Relative Quantification of Tissue Carbonylome.

Carbonylation is a nonenzymatic irreversible posttranslational protein modification and the main hallmark of protein oxidative damage. Elevated levels of protein carbonyl groups have been detected in age-related and metabolic diseases such as obesity, diabetes, Alzheimer, Parkinson, and several other oxidative stress-related maladies. Interestingly, many studies have shown that only a subset of proteins is carbonylated under the conditions of oxidative stress, demonstrating that carbonylation is a highly selective process. As a consequence, identifying and quantifying the disease-induced changes on a certain carbonylome are crucial to understanding the etiology and progression of numerous diseases and then designing adequate prevention/palliation strategies. However, the low abundance of carbonylated proteins in vivo, the enormous diversity of reactive species, and their relative lability make the analysis of carbonylated proteins a challenging task for redox proteomic technology. Therefore, we present a proteomic approach based on the labeling of carbonyls formed in vivo on proteins using the fluorescein 5-thiosemicarbazide (FTSC) tag to detect the subset of carbonylated proteins among a complex mixture of proteins regardless of the nature of carbonyl adduct, isolation and relative quantification of carbonylated proteins in 2D gel electrophoresis, and protein identification by LC-MS/MS analysis. This method has been successfully used for the evaluation of in vivo protein carbonylation in very diverse animal tissues (plasma, liver, kidney, skeletal muscle, and adipose tissue) and species (from fish to mammalian) and has also been applied in different research fields (from food technology to nutrition), demonstrating its robustness and reliability.

Gender influence on antioxidant capacity and oxidative damage in saliva of children with autism spectrum disorder: a preliminary study

Abstract Introduction Autism is a complex neurological disorder with multifactorial causes, which compromises physical, psychological, emotional and social skills. This disorder also impairs dental care and patient oral health. Saliva can be easily obtained using a non-invasive technique and has been used in the diagnosis and screening of many pathologies. Total antioxidant capacity of saliva is reduced in the presence of caries in children and has been studied as a possible marker for autism. However, data on salivary antioxidants in children with autism spectrum disorder (ASD) is scarce. Objective This preliminary study evaluated the existence of gender influence on non-enzymatic antioxidant capacity and protein oxidative damage in the saliva of children with this disorder. Our hypothesis is that there are gender differences in these biochemical parameters in children with austim. Material and method Whole saliva samples were collected by aspiration between 8:00-11:00 am from 12 girls and 12 boys aged 5 to 15 years, with previous diagnosis for this disorder. Total antioxidant capacity, uric acid, protein concentration and oxidative damage to proteins were analyzed by spectrophotometry. Values were presented as mean ± standard deviation and tested for adherence to normality (Shapiro-Wilk test). The data were then compared using the Mann-Whitney test, considering a 5% significance level. Result There was no significant difference between groups for all analyzed parameters (P>0.05). Conclusion Based on the results obtained, we concluded that gender does not influence the levels of oxidative stress markers in the saliva of children with ASD.

Inhibition of Protein Glycation and OH <sup>•</sup>-Induced Protein Fragmentation by Fenugreek Phytochemicals

Glycation of proteins and generation of advanced glycation end products (AGEs) are the fundamental causes in the development of oxidative stress-induced protein modification and diabetes complications. The hyperglycemic condition causes non-enzymatic protein glycation, initiated by the reaction between the carbonyl groups of sugars with the amino groups of proteins forming the amadori products. Amadori products induce further oxidation, generating dicarbonyl compounds to form cross-linking fluorescent and non-fluorescent adduct called AGEs. In this study, the protective effect of fenugreek phytochemicals on protein glycation and oxidation-induced protein fragmentation was evaluated. Fenugreek phytochemicals (phenolics and alkaloids) protected oxidation-induced protein fragmentation and inhibited protein glycation. They also prevented the formation of fluorescent and non-fluorescent AGEs, and glycation-mediated protein conformational changes. Fenugreek phytochemicals effectively scavenged reactive carbonyl intermediates and decreased protein thiol modification. Collectively, these results suggest the potential utility of fenugreek phytochemicals as functional food ingredients for the control of diabetes and its complications. Funding Information: This work is financially supported by the planning commission and Council of Scientific and Industrial Research, Govt. India, as part of 12th- five-year plan project (BSC-0404). Declaration of Interests: The authors declare that they have no competing interests.

Hepato-nephroprotective Role of Lepidium sativum against Oxidative Stress Induced by Dexamethasone in Rats

The current research was prepared to explore the hepato-nephroprotective effect of Lepidium sativum seedaquatic extract against oxidative stress stimulated by dexamethasone in rats. Animals were classified asfollows; group one was used as control while groups 2, 3 and 4 were treated orally with Lepidium sativumseed extract (LSSE; 20 mg/kg, daily), interperitoneally with dexamethasone (DEX; 1 mg/kg BW) andLSSE plus DEX for 14 days, respectively. Administration of DEX elevated thiobarbituric acid reactivesubstances (TBARS), hydrogen peroxide (H2O2), and kidney and liver function biomarkers level, andlactate dehydrogenase activity. While enzymatic (SOD, CAT, GPx, GR, GST) and non-enzymatic (GSH)antioxidants, protein content, and alkaline phosphatase activity were significantly decreased. Otherwise, ratssupplemented with LSSE singly declined lipid peroxidation and improved most of the studied parameters.Moreover, rats pretreated with LSSE then received DEX showed significant alleviation in lipid peroxidation,antioxidant status and biochemical indices with respect to DEX treated group. Conclusively, LSSE hasbeneficial protective effects and has the capability to counteract the harmful influence of DEX. So, Lepidiumsativum might represent a novel approach in the therapy of dexamethasone because of its antioxidantproperties.

Factor VIII and Factor V Membrane Bound Complexes.

The formation of membrane-bound complexes between specific coagulation factors at different cell surfaces is required for effective blood clotting. The most important of these complexes, the intrinsic Tenase and Prothrombinase complexes, are formed on the activated platelet surface during the propagation phase of coagulation. These two complexes are highly specific in their assembly mechanism and function modulated by anionic membranes, thus offering desirable targets for pharmaceutical interventions. Factor V (FV) and factor VIII (FVIII) are highly homologous non-enzymatic proteins. In their active state, FVa and FVIIIa serve as cofactors for the respective serine proteases factor Xa (FXa) and factor IXa (FIXa), significantly increasing their catalytic activity. This is achieved by forming well organized membrane-bound complexes at the phosphatidylserine rich activated platelet membrane in the presence of Ca2+ ions. The tenase (FVIIIa/FIXa) complex, catalyzes the proteolytic conversion of FX to FXa. Subsequently the prothrombinase (FVa/FXa) complex catalyzes the conversion of prothrombin to thrombin, required for efficient blood clotting. Although significant knowledge of FV and FVIII biochemistry and regulation has been achieved, the molecular mechanisms of their function are yet to be defined. Understanding the geometric assembly of the tenase and prothrombinase complexes is paramount in defining the structural basis of bleeding and thrombotic disorders. Such knowledge will enable the design of efficient pro- and anticoagulant therapies critical for regulating abnormal hemostasis. In this chapter, we will summarize the findings to date, showing our achievement in the field and outlining the future findings required to grasp the complexity of these proteins.

Potential Role of Platelet-Activating C-Type Lectin-Like Proteins in Viper Envenomation Induced Thrombotic Microangiopathy Symptom.

Envenomation by viperid snakes may lead to severe bleeding, consumption coagulopathy, and thrombotic microangiopathy symptoms. The exact etiology or toxins responsible for thrombotic microangiopathy symptoms after snake envenomation remain obscure. Snake C-type lectin-like proteins (snaclecs) are one of the main non-enzymatic protein constituents in viper venoms, of which a majority are considered as modulators of thrombosis and hemostasis. In this study, we demonstrated that two snaclecs (mucetin and stejnulxin), isolated and identified from Protobothrops mucrosquamatus and Trimeresurus stejnegeri venoms, directly induced platelet degranulation and clot-retraction in vitro, and microvascular thrombosis has been confirmed in various organs in vivo. These snaclecs reduced cerebral blood flow and impaired motor balance and spatial memories in mice, which partially represent the thrombotic microangiopathy symptoms in some snakebite patients. The functional blocking of these snaclecs with antibodies alleviated the viper venom induced platelet activation and thrombotic microangiopathy-like symptoms. Understanding the pathophysiology of thrombotic microangiopathy associated with snake envenoming may lead to emerging therapeutic strategies.

Is the Oxidative Stress in Obstructive Sleep Apnea Associated With Cardiovascular Complications?-Systematic Review.

Obstructive sleep apnea (OSA) is a prevalent, underdiagnosed disease and is considered an independent risk factor for cardiovascular disease. The exact mechanism of cardiovascular complications (CVC) development as a complication of OSA is not entirely understood. Oxidative stress is suspected to be the essential factor in initiating various comorbidities in OSA. Biomarkers of nonenzymatic lipid and protein peroxidation, DNA repair and antioxidant capabilities measured in serum, plasma and urine are frequently used to assess the presence of oxidative stress. We conducted a systematic review and quality assessment of available observational analytic studies to determine whether there is an association between oxidative stress and OSA in patients with prevalent CV disease compared to (a) patients with prevalent CV disease but no OSA, (b) patients with prevalent CV disease and less severe OSA and (c) patients with OSA and no overt CV disease. This systematic review demonstrated that, while oxidative stress is associated with OSA, there was no clear difference in the severity of oxidative stress between OSA patients with or without cardiovascular complications.

Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins represents a major pharmacologic challenge. The clinical success of thalidomide analogs demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1–3, but a significant subset of proteins are recalcitrant to targeted protein degradation using current approaches4,5. Here we report an alternative mechanism, whereby a small molecule induces highly specific, reversible polymerization, sequestration into cellular foci, and subsequent degradation of a target protein. BI-3802 is a small molecule that binds the BTB domain of the oncogenic transcription factor BCL6 and results in proteasomal degradation6. We used cryo-EM to reveal how the solvent-exposed moiety of a BCL6 inhibitor contributes to a composite ligand/protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to superior pharmacological activity. These findings create new avenues for the development of therapeutics and synthetic biology.

Self-Immolative Difluorophenyl Ester Linker for Affinity-Based Fluorescence Turn-on Protein Detection.

Currently most fluorogenic probes are developed for the analysis of enzymes, where a bond breaking or rearrangement reaction is required to transform a nonfluorescent enzymatic substrate into a fluorescent product. However, this approach cannot be used for proteins that do not possess enzymatic activities. In this article, we show that fluorogenic probes with a self-immolative difluorophenyl ester linker can mimic the bond disassembly processes of fluorogenic enzyme substrates for the rapid analysis of nonenzymatic proteins. Although numerous self-immolative reagents have shown promising applications in sensors, drug delivery systems, and material chemistry, all of them are triggered by either enzymes or small reactive molecules. In our strategy, the probe binds to the protein via a specific protein-ligand interaction, inducing a chemical reaction between the self-immolative linker and an amino acid of the protein, thereby triggering a cascade reaction that leads to the activation and release of the fluorogenic reporter. In contrast, a phenyl ester linker without the difluoro substituent cannot be triggered to release the fluorogenic reporter. With this probe design, live-cell imaging of extracellular and intracellular endogenous tumor marker proteins can be achieved with high selectivity and sensitivity.

SEIPIN: A Key Factor for Nuclear Lipid Droplet Generation and Lipid Homeostasis.

Lipid homeostasis is essential for normal cell physiology. Generally, lipids are stored in a lipid droplet (LD), a ubiquitous organelle consisting of a neutral lipid core and a single layer of phospholipid membrane. It is thought that LDs are generated from the endoplasmic reticulum and then released into the cytosol. Recent studies indicate that LDs can exist in the nucleus, where they play an important role in the maintenance of cell phospholipid homeostasis. However, the details of nuclear lipid droplet (nLD) generation have not yet been clearly characterized. SEIPIN is a nonenzymatic protein encoded by the Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene. It is associated with lipodystrophy diseases. Many recent studies have indicated that SEIPIN is essential for LDs generation. Here, we review much of this research in an attempt to explain the role of SEIPIN in nLD generation. From an integrative perspective, we conclude by proposing a theoretical model to explain how SEIPIN might participate in maintaining homeostasis of lipid metabolism.

Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution.

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three β-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.

Nephroprotective role of bromelain against oxidative injury induced by aluminium in rats

The present study was designed to investigate the nephroprotective effect of bromelain against oxidative stress stimulated by aluminium chloride in rats. Rats were grouped as follows; group one was used as control while groups 2, 3 and 4 were treated orally with bromelain (250 mg/kg, daily), aluminium chloride (AlCl3; 34 mg/kg BW, every other day) and bromelain plus AlCl3 for 30 days, respectively. Administration of AlCl3 caused a significant reduction in rats’ body and kidney weights, and increased Al accumulation in kidney tissue. Also, AlCl3 treatment elevated thiobarbituric acid reactive substances, hydrogen peroxide, kidney functions biomarkers levels and lactate dehydrogenase activity. While enzymatic (SOD, CAT, GPx, GR, GST) and non-enzymatic (GSH) antioxidants, protein content, and alkaline phosphatase activity were significantly decreased. In addition, significant alterations in lipid and protein profiles were detected. Furthermore, histopathological and immunohistochemical variations were seen in kidney sections supporting the obtained biochemical changes. Otherwise, rats supplemented with bromelain singly declined lipid peroxidation and improved most of the studied parameters. Moreover, rats pretreated with bromelain followed by AlCl3 intoxication showed significant alleviation in lipid peroxidation, antioxidant status and biochemical indices with respect to AlCl3 treated group. Conclusively, bromelain has beneficial protective effects and has the capability to counteract the toxic influence of AlCl3. So, bromelain might represent a novel approach in the therapy of metal toxicity because of its antioxidant and chelating properties.

Non-Enzymatic Protein Acetylation by 7-Acetoxy-4-Methylcoumarin: Implications in Protein Biochemistry.

The semi-synthetic acetoxycoumarins are known to acetylate proteins using novel enzymatic Calreticulin Transacetylase (CRTAase) system in cells. However, the nonenzymatic protein acetylation by polyphenolic acetates is not known. To investigate the ability of 7-acetoxy-4-methyl coumarin (7-AMC) to acetylate proteins non-enzymatically in the test tube. We incubated 7-AMC with BSA and analyzed the protein acetylation using Western blot technique. Further, BSA induced biophysical changes in the spectroscopic properties of 7-AMC was analyzed using Fluorescence spectroscopy. Using pan anti-acetyl lysine antibody, herein we demonstrate that 7-AMC acetylates Bovine Serum Albumin (BSA) in time and concentration dependent manner in the absence of any enzyme. 7-AMC is a relatively less fluorescent molecule compared to the parental compound, 7- hydroxy-4-methylcoumarin (7-HMC), however the fluorescence of 7-AMC increased by two fold on incubation with BSA, depending on the time of incubation and concentration of BSA. Analysis of the reaction mixture of 7-AMC and BSA after filtration revealed that the increased fluorescence is associated with the compound of lower molecular weight in the filtrate and not residual BSA, suggesting that the less fluorescent 7-AMC undergoes self-hydrolysis in the presence of protein to give highly fluorescent parental molecule 7-HMC and acetate ion in polar solvent (phosphate buffered saline, PBS). The protein augmented conversion of 7-AMC to 7-HMC was found to be linearly related to the protein concentration. Thus protein acetylation induced by 7-AMC could also be non-enzymatic in nature and this molecule can be exploited for quantification of proteins.

Effect of Glyoxal Modification on a Critical Arginine Residue (Arg-31α) of Hemoglobin: Physiological Implications of Advanced Glycated end Product an in vitro Study.

Non-enzymatic protein glycation is involved in structure and stability changes that impair protein functionality, resulting in several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer's disease, Parkinson's disease and Andrade's syndrome). Glyoxal, an endogenous reactive oxoaldehyde, increases in diabetes and reacts with several proteins to form advanced glycation end products through Maillard-like reaction. Human hemoglobin, the most abundant protein in blood cells is subjected to nonenzymatic modification by reactive oxoaldehydes in diabetic condition. In the present study, the effect of a low concentration of glyoxal (5 μM) on hemoglobin (10 μM) has been investigated following a period of 30 days incubation in vitro. Different techniques, mostly biophysical and spectroscopic (e.g. circular dichroism, differential scanning calorimetric study, dynamic light scattering, mass spectrometry, etc.) were used to study glyoxal-induced changes of hemoglobin. Glyoxal-treated hemoglobin exhibits decreased absorbance around 280 nm, decreased fluorescence and reduced surface hydrophobicity compared to normal hemoglobin. Glyoxal treatment enhances the stability of hemoglobin and lowers its susceptibility to thermal aggregation compared to control hemoglobin as seen by different studies. Finally, peptide mass fingerprinting study showed glyoxal to modify an arginine residue of α-chain of hemoglobin (Arg-31α) to hydroimidazolone. Increased level of glyoxal in diabetes mellitus as well as its high reactivity may cause modifications of the heme protein. Thus, considering the significance of glyoxal-induced protein modification under physiological conditions, the observation appears clinically relevant in terms of understanding hydroimidazolone-mediated protein modification under in vivo conditions.

Form of Silica Improves Yield, Fruit Quality and Antioxidant Defense System of Tomato Plants under Salt Stress

Tomato crop is valuable worldwide thanks to its commercial and nutritional value, which plays a very important role in the human diet. However, in arid areas, tomato crops can be found with high salt content. Salinity is a major problem for agriculture, as it decreases productivity, lowers economic yields, and induces soil erosion. The application of silicon has been observed to increase tolerance to abiotic stress and specifically to salt stress. Therefore, the aim of this study is to evaluate the application of K2SiO3 and SiO2 nanoparticles (SiO2 NPs) on the growth, antioxidant content, and tolerance to saline stress of tomato plants. Plant growth, fruit quality parameters (pH, titratable acidity, total soluble solids, firmness), antioxidant capacity (ABTS, DPPH), enzymatic (SOD, PAL, APX, CAT, GPX) and non-enzymatic (flavonoids, phenols, vitamin C, β-carotene, lycopene) antioxidant compounds, chlorophylls, proteins, and H2O2 were evaluated. The application of SiO2 NPs at 500 mg L−1 had positive effects on the plants that were not subjected to stress, increasing the average fruit weight, fruit yield, and chlorophyll, phenol, glutathione, and GPX activity. Meanwhile, in plants under salt stress, it helped to maintain the concentration of chlorophylls, GSH, PAL activity, and vitamin C. The application of SiO2 NPs is more effective than K2SiO3 at inducing positive responses in tomato plants subjected to stress by NaCl.