Inactivation Of Enzyme Research Articles

Alkyne‐ and azide‐functionalised cyclophellitol derivatives for selective glucuronidase targeting

Heparanase is an endo‐acting β‐glucuronidase and the only known enzyme responsible for the regulation of heparan sulfate structures in the extracellular matrix. The enzyme is found to be significantly upregulated in aggressive cancer types, aiding cell proliferation by liberation of growth factors, increasing angiogenesis and metastasis. Despite much interest in the development of inhibitors to control this activity, no compound targeting heparanase has yet reached the clinic. While mechanism‐based inhibitors derived from the epoxide carbocycle cyclophellitol are very potent enzyme inactivators, one challenge in their development is in achieving selectivity for heparanase over related glycosidases. We here present the synthesis of three cyclophellitol scaffolds presenting azide and alkyne handles at key positions, which are amenable to facile elaboration via copper‐catalysed azide‐alkyne cycloadditions to aid in exploring the structure‐activity relationship for selective inhibitors of heparanase.

Disruptions of rpiAB Genes Encoding Ribose-5-Phosphate Isomerases in E. coli Increases Sensitivity of Bacteria to Antibiotics.

In Escherichia coli cells, the main enzymes involved in pentose interconversion are ribose-5-phosphate isomerases RpiA and RpiB and ribulose-5-phosphate epimerase Rpe. The inactivation of rpiAB limits ribose-5-phosphate (R5P) synthesis via the oxidative branch of the pentose phosphate pathway (PPP) and unexpectedly results in antibiotic supersensitivity. This type of metabolism is accompanied by significant changes in the level of reducing equivalents of NADPH and glutathione, as well as a sharp drop in the ATP pool. However, this redox and energy imbalance does not lead to the activation of the soxRS oxidative stress defense system but the increased sensitivity to oxidants paraquat and H2O2. The deletion of rpiAB leads to a significant increase in the activity of transketalase (Tkt), a key enzyme of the nonoxidative branch of the PPP and increased sensitivity to ribose added in the growth medium. The phenotype of supersensitivity of rpiAB to antibiotics and ribose can be suppressed by activating the utilization of sedoheptulose-7-phosphate, which originates from R5P, to LPS synthesis or limitation of nucleoside catabolism by the inactivation of the DeoB enzyme, responsible for conversion of ribose-1-phospate to R5P. Our results indicate that the induction of unidirectional synthesis of R5P is the cause of supersensitivity to antibiotics in rpiAB mutant.

Impact of accelerated aging on seed quality, seed coat physical structure and antioxidant enzyme activity of Maize (Zea mays L.).

Aging induces many deteriorative changes to seeds during storage like protein degradation, enzyme inactivation and loss of membrane integrity. In this study, we investigate the impact of accelerated aging on seed quality, seed coat physical structure and antioxidant enzyme activity of maize. Three genotypes African Tall, MAH 14-5 and a local landrace were selected and artificially aged for 96 and 120 h. The aging process led to a decrease in germination, vigour, and total dehydrogenase in seeds, while the electrical conductivity of seed leachates increased, indicating a decline in seed quality. Additionally, there was a variation in the microsculpture pattern of seed coats between genotypes. There was an accumulation of damage on the seed coat surface as the seeds aged and higher damage occurred in African Tall followed by MAH 14-5 and local landrace. Higher catalase, superoxide dismutase, peroxidase and polyphenol oxidase activity were reported in the seed coat of Local landrace and MAH 14-5 that resisted aging and deterioration while, lower catalase, superoxide dismutase, peroxidase and polyphenol oxidase activity was reported in African Tall seed coat that deteriorated during aging. Decrease antioxidant activity in aged seeds might be a possible cause of seed deterioration due to the accumulation of free radicals. Thus, these results clearly show the influence of seed coat structure and antioxidant activity on seed quality during aging.

EXTH-23. NEW DNA-CROSSLINKING CHEMOTHERAPY IS EFFECTIVE AGAINST POST-TEMOZOLOMIDE MISMATCH REPAIR-DEFICIENT PATIENT-DERIVED HYPERMUTANT GLIOMAS

Abstract The DNA mismatch repair (MMR) pathway triggers glioblastoma (GBM) apoptosis when base mismatches induced by the alkylating agent temozolomide (TMZ) cannot be repaired. GBMs often develop hypermutation and resistance to TMZ through acquired inactivation of DNA MMR enzymes (MSH2, MSH6, MLH1, PMS2). A newly developed therapeutic, KL-50, fluoro-ethylates DNA bases, leading to DNA inter-strand cross-links. This form of DNA damage triggers MMR-independent apoptosis. Thus, KL-50 can target MMR-deficient tumor cells. Previous work showed that KL-50 is effective against GBM cell lines that were rendered TMZ-resistant through shRNA knockdown of MMR genes (PMID 35901163). In those studies, KL-50 was most effective when used in combination with an MGMT enzyme inhibitor, or in MGMT-deficient cells. Here, we extend those findings with a preclinical trial of KL-50 in GBM patient-derived xenografts (PDX), including PDX models of post-TMZ GBM that acquired MMR mutations and hypermutation through serial TMZ exposure (PMID 31852831). Among TMZ-naïve PDX, KL-50 extended survival of engrafted mice by 24 days for GBM6 (partially MGMT-deficient), and by 38 days for GBM12 PDX (fully MGMT-deficient) compared to vehicle (p<0.0001, Log-Rank). Combining KL-50 with 4.0 Gy radiation further extended survival of GBM12 mice (9 days, p=0.02), but not GBM6 mice. With engraftments of GBM6R-m185, a post-TMZ, MMR-deficient derivative of GBM6, 14/14 (100%) of KL-50 treated mice are still alive 60 days post-engraftment compared to 0/13 (0%) of vehicle control mice (median survival 37 days, p<0.0001, experiment ongoing). Complementary in vitro studies showed that GBM6R-m185 cells are more sensitive to KL-50 than TMZ at matched concentrations of 50µM (p=0.001, one-way ANOVA) and 100µM (p=0.047). These data demonstrate the potential of KL-50 in treating post-TMZ MMR-deficient recurrent gliomas.

Expanding the antiprotozoal activity and the mechanism of action of n-butyl and iso-butyl ester of quinoxaline-1,4-di-N-oxide derivatives against Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica. An in vitro and in silico approach

In this study, n-butyl and iso-butyl quinoxaline-7-carboxylate-1,4-di-N-oxide derivatives were evaluated in vitro against Giardia lamblia (G. lamblia), Trichomonas vaginalis (T. vaginalis), and Entamoeba histolytica (E. histolytica). The potential mechanism of action determination was approached by in silico analysis on G. lamblia and T. vaginalis triosephosphate isomerase (GlTIM and TvTIM, respectively), and on E. histolytica thioredoxin reductase (EhTrxR). Enzyme inactivation assays were performed on recombinant GlTIM and EhTrxR. Compound T-167 showed the best giardicidal activity (IC50 = 25.53 nM) and the highest inactivation efficiency against GlTIM without significantly perturbing its human homolog. Compounds T-142 and T-143 showed the best amoebicidal (IC50 = 9.20 nM) and trichomonacidal (IC50 = 45.20 nM) activity, respectively. Additionally, T-143 had a high activity as giardicial (IC50 = 29.13 nM) and amoebicidal (IC50 = 15.14 nM), proposing it as a broad-spectrum antiparasitic agent. Compounds T-145, and T-161 were the best EhTrxR inhibitors with IC50 of 16 µM, and 18 µM, respectively.

Methylglyoxal-Induced Modifications in Human Triosephosphate Isomerase: Structural and Functional Repercussions of Specific Mutations.

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme's structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI's susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders.

Interplay between de novo and salvage pathways of GDP-fucose synthesis

GDP-fucose is synthesised via two pathways: de novo and salvage. The first uses GDP-mannose as a substrate, and the second uses free fucose. To date, these pathways have been considered to work separately and not to have an influence on each other. We report the mutual response of the de novo and salvage pathways to the lack of enzymes from a particular route of GDP-fucose synthesis. We detected different efficiencies of GDP-fucose and fucosylated structure synthesis after a single inactivation of enzymes of the de novo pathway. Our study demonstrated the unequal influence of the salvage enzymes on the production of GDP-fucose by enzymes of the de novo biosynthesis pathway. Simultaneously, we detected an elevated level of one of the enzymes of the de novo pathway in the cell line lacking the enzyme of the salvage biosynthesis pathway. Additionally, we identified dissimilarities in fucose uptake between cells lacking TSTA3 and GMDS proteins.

Noncovalent Inhibition and Covalent Inactivation of Proline Dehydrogenase by Analogs of N-Propargylglycine.

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ1-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. N-propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH. This compound consists of a recognition module (glycine) that directs the inactivator to the active site and an alkyne warhead that reacts with the FAD after oxidative activation, leading to covalent modification of the FAD N5 atom. Here we report structural and kinetic data on analogs of N-propargylglycine with the goals of understanding the initial docking step of the inactivation mechanism and to test the allyl group as a warhead. The crystal structures of PRODH complexed with unreactive analogs in which N is replaced by S show how the recognition module mimics the substrate proline by forming ion pairs with conserved arginine and lysine residues. Further, the C atom adjacent to the alkyne warhead is optimally positioned for hydride transfer to the FAD, providing the structural basis for the first bond-breaking step of the inactivation mechanism. The structures also suggest new strategies for designing improved N-propargylglycine analogs. N-allylglycine, which consists of a glycine recognition module and allyl warhead, is shown to be a covalent inactivator; however, it is less efficient than N-propargylglycine in both enzyme inactivation and cellular assays. Crystal structures of the N-allylglycine-inactivated enzyme are consistent with covalent modification of the N5 by propanal.

Bioinformatic Analysis of Oxygen Sensitivity in Arcobacter bivalviorum's Pyruvate:Ferredoxin Oxidoreductase (PFOR)

Background: Arcobacter bivalviorum is an emerging foodborne pathogen particularly found in shellfish and other bivalves. This bacterium's metabolic versatility is highlighted by its possession of both the Pyruvate Dehydrogenase Complex (PDC) and Pyruvate:Ferredoxin Oxidoreductase (PFOR). The PFOR enzyme is crucial for linking glycolysis to the citric acid cycle. Previous studies of PFOR enzyme reveal its susceptibility to oxygen damage due to its reliance on Fe-S clusters for electron transfer.Methods: The current study employs bioinformatic approaches to explore the oxygen sensitivity of A. bivalviorum's PFOR, comparing it with Desulfovibrio africanus and Moorella thermoacetica. The UniProt database was used to obtain the sequences of PFOR from D. africanus (CAA70873), A. bivalviorum (AXH11209), and M. thermoacetica (Q2RMD6). This comparative analysis sheds light on the structural similarities and differences between the enzymes, providing a deeper understanding of their functional mechanisms.Result: PFOR enzyme is a heterodimer, with its functional subunits containing three [4Fe-4S]²⁺ clusters. Its exposed Fe-S clusters are vulnerable to oxygen and ROS, leading to enzyme inactivation. A comparison of PFOR sequences from D. africanus, M. thermoacetica, and A. bivalviorum reveals a crucial difference: the final 43 residues at the C-terminal of D. africanus are missing in the other two enzymes, depriving the M. thermoacetica and A. bivalviorum enzymes of a self-protective mechanism.Conclusion: Pyruvate:ferredoxin oxidoreductase (PFOR) in A. bivalviorum lacks protection from oxidative damage due to the absence of the final 43 amino acids at the C-terminal.Keywords: PFOR; Homology modeling; oxygen sensitivity; Foodborne pathogen

A Dynamic Loop in Halohydrin Dehalogenase HheG Regulates Activity and Enantioselectivity in Epoxide Ring Opening.

Halohydrin dehalogenase HheG and its homologues are remarkable enzymes for the efficient ring opening of sterically demanding internal epoxides using a variety of nucleophiles. The enantioselectivity of the respective wild-type enzymes, however, is usually insufficient for application and frequently requires improvement by protein engineering. We herein demonstrate that the highly flexible N-terminal loop of HheG, comprising residues 39 to 47, has a tremendous impact on the activity as well as enantioselectivity of this enzyme in the ring opening of structurally diverse epoxide substrates. Thus, highly active and enantioselective HheG variants could be accessed through targeted engineering of this loop. In this regard, variant M45F displayed almost 10-fold higher specific activity than wild type in the azidolysis of cyclohexene oxide, yielding the corresponding product (1S,2S)-2-azidocyclohexan-1-ol in 96%eeP (in comparison to 49%eeP for HheG wild type). Moreover, this variant was also improved regarding activity and enantioselectivity in the ring opening of cyclohexene oxide with other nucleophiles, demonstrating even inverted enantioselectivity with cyanide and cyanate. In contrast, a complete loop deletion yielded an inactive enzyme. Concomitant computational analyses of HheG M45F in comparison to wild type enzyme revealed that mutation M45F promotes the productive binding of cyclohexene oxide and azide in the active site by establishing noncovalent C-H ··π interactions between epoxide and F45. These interactions further position one of the two carbon atoms of the epoxide ring closer to the azide, resulting in higher enantioselectivity. Additionally, stable and enantioselective cross-linked enzyme crystals of HheG M45F were successfully generated after combination with mutation D114C. Overall, our study highlights that a highly flexible loop in HheG governs the enzyme's activity and selectivity in epoxide ring opening and should thus be considered in future protein engineering campaigns of HheG.

Effect of pulsed light on enzyme inactivation, colour, firmness, surface morphology, and bioactive compounds in fresh and dried whole red chilies (Capsicum annuum var. longum)

The effect of pulsed light on enzymatic activity and biochemical stability of red chillies was investigated. Balancing and reducing the aw and moisture content to 0.6 aw and 0.35 aw is crucial to attain biochemical stability. Upon drying at 60 °C/5 h + 40 °C/2 h and 70 °C/11 h + 55 °C/4 h + 40 °C/2 h the enzymes inactivated to more than 30 %, respectively. Hence, to completely inactivate the enzymes, a technology has been introduced i.e., pulsed light treatment (PLT), which employed 0.53–2.59 J·cm−2. The stability of red chillies was evaluated based on the quality attributes such as enzyme activity, total phenolics, flavonoids, antioxidant capacity, ascorbic acid, capsiacinoids, and carotenoids. After treatment at fluence of 2.59 J·cm−2, there was >90 % inactivation of PPO and POD. Based on kinetic modelling, it is showed PLT better preserved the colour, texture, morphology, and bioactive compounds in 0.6 and 0.35 aw than 0.85 aw red chillies. Morphological studies determined the changes in red chillies after PLT. FTIR and GCMS analysis revealed the structural and compositional changes occurring after PLT. Therefore, drying combined with PL can be a potential alternative method to blanching along with maximizing the availability of bioactives.

Lipase Substrate Anchoring Dynamics Guided the Rational Design of Novel Deep Eutectic Solvents for Glucose Ester Synthesis.

Glucose esters, heralded for their emulsifying and solubilizing properties, have found increasing application in food, pharmaceutical, and cosmetic sectors. However, the environmental impact of chemical synthesis and the problem of enzyme inactivation in enzymatic synthesis have hindered their application. To this end, deep eutectic solvents (DESs) with stabilized enzyme activity and better solubility properties are considered a promising solution. In the study, 12 kinds of hydrophilic DESs and 22 kinds of hydrophobic DESs were screened, and we examined their efficiency in the synthesis of glucose esters. The results indicated that d,l-menthol-based DESs have excellent performance, with the highest yield of 92.85%. The molecular dynamics simulations suggested that it can be attributed to DESs changing the size of substrate binding pocket, which in turn affects the substrate anchoring ability and the catalytic efficiency of lipase. To further evaluate the effects of the solvent on the substrate anchoring ability of enzymes, a novel strategy, namely, substrate anchoring index, which is based on the stability of the tetrahedral intermediate, was proposed and used for the designing of more efficient DESs. Fortunately, novel DESs based on cyclohexanone (menthol analogues) were successfully developed with a yield of 98.85%.

High-Temperature Short-Time and Ultra-High-Temperature Processing of Juices, Nectars and Beverages: Influences on Enzyme, Microbial Inactivation and Retention of Bioactive Compounds

HTST (high-temperature short-time) pasteurization and UHT (ultra-high-temperature) sterilization are techniques commonly used in the dairy industry. Although the use of these methods in fruit and vegetable processing is also well known, the multitude of diverse food matrices determines the need to test and adjust process parameters in order to obtain the best quality of the final product. HTST and UHT are methods that provide effective inactivation of microorganisms and enzymes. Despite the fact that UHT and HTST are thermal processes that cause degradation of bioactive ingredients or color change, in many cases, these two methods are superior to traditional pasteurization, which uses significantly longer exposures to high temperatures. Therefore, this article aims to review the effect of HTST and UHT processing on the quality of juices, nectars and beverages, taking into consideration the quality characteristics, like the presence of microorganisms, pH, titratable acidity, total soluble solids, turbidity, color parameters, contents of bioactive components, antioxidant activity, enzymatic activity and volatile compounds. The impacts of HTST and UHT methods on various food products are discussed, including the food matrix, preservation parameters and the mechanism of interaction. The ability to modify the processing parameters can allow for the selection of adequate preservation parameters for individual products and better results than other unconventional methods, such as HPP (high-pressure processing) or PEF (pulsed electric field). Based on the cited literature, it can be concluded that pH, titratable acidity and TSS most often experience slight changes. As for the other parameters considered, it is extremely important to choose the right temperature and duration for a specific food matrix.

Activity and phosphatidylcholine transfer protein interactions of skeletal muscle thioesterase Them2 enable hepatic steatosis and insulin resistance

Thioesterase superfamily member 2 (Them2), a long-chain fatty acyl-CoA thioesterase that is highly expressed in oxidative tissues, interacts with phosphatidylcholine transfer protein (PC-TP) to regulate hepatic lipid and glucose metabolism and to suppress insulin signaling. High-fat diet (HFD)-fed mice lacking Them2 globally or specifically in skeletal muscle, but not liver, exhibit reduced hepatic steatosis and insulin resistance. Here, we report that the capacity of Them2 in skeletal muscle to promote hepatic steatosis and insulin resistance depends on both its catalytic activity and interaction with PC-TP. Two residues of Them2 catalytic site were mutated (N50A/D65A) to produce the inactive enzyme while maintaining its homotetrameric structure and interaction with PC-TP. Restoration of skeletal muscle expression in Them2-/- mice using recombinant adeno-associated virus revealed that wild-type (WT), but not N50A/D65A Them2, promoted HFD-induced weight gain and hepatic steatosis. This was accompanied by greater impairment of insulin sensitivity in WT compared with N50A/D65A Them2. Pharmacological inhibition or genetic ablation of PC-TP attenuated these effects. In reductionist experiments, conditioned medium collected from WT primary cultured myotubes promoted excess lipid accumulation in oleic acid-treated primary cultured hepatocytes relative to Them2-/- myotubes, which was attributable to secreted extracellular vesicles (EV). Reconstitution of Them2 expression in Them2-/- myotubes affirmed the requirements for catalytic activity and PC-TP interactions for EV to promote lipid accumulation in hepatocytes. These studies provide valuable mechanistic insights whereby Them2 in skeletal muscle promotes hepatic steatosis and establish both Them2 and PC-TP as represent attractive targets for managing metabolic dysfunction-associated steatotic liver disease.

UV‐A Light Dehydration: Kinetics of Microbial Inactivation Against Gram‐Positive and Gram‐Negative Bacteria

ABSTRACTUV‐A light exposure (365 nm, 4.6 ± 0.2 mW/cm2) was combined with low relative humidity (RH) air flow at room temperature to dehydrate sweet potatoes and reduce the population of inoculated bacteria. Control samples underwent dehydration with low RH air at room temperature and in the absence of UV‐A light to assess the importance of UV‐A light in the dehydration and microbial reduction processes. The UV‐A light‐dehydrated sweet potatoes resulted in the removal of approximately 97.2% ± 2% of the original mass of water, which was significantly higher than the control samples. Infrared spectroscopy analyses confirmed the preservation of the physical and chemical integrity of the UV‐A light‐dehydrated samples. Despite the absence of pretreatments for enzyme inactivation, the UV‐A light‐dehydrated sweet potato did not exhibit a decrease in luminosity or darkening of color often associated with dehydration. Additionally, the utilization of UV‐A light for the dehydration of sweet potatoes inoculated with ~6 log(CFU/gDry solids) of Escherichia coli K12 resulted in a 99.9% ± 0.1% or 3.1 ± 0.5 log(CFU/gDry solids) reduction with only a 92.2% ± 0.1% or 1.3 ± 0.5 log(CFU/gDry solids) reduction resulting from the control samples dehydrated without UV‐A exposure. In the case of samples inoculated with ~6 log(CFU/gDry solids) of Listeria innocua L2 there was a 99.2% ± 0.5% or 2.2 ± 0.3 log(CFU/gDry solids) reduction when UV‐A light was utilized and only a 60.9% ± 10.3% or 0.4 ± 0.1 log(CFU/gDry solids) reduction when samples were dehydrated in its absence.

Current status of technological advancement of ultrasound processing in the food industry and its SWOT analysis.

The use of conventional food processing techniques has almost vanished due to increase in demand with respect to time, thus opening new avenues for emerging technologies. Ultrasound (US) is a rapid, multifaceted, promising, and noninvasive green technology. It has attracted the attention of both industrial experts and scientists for its probable use in food processing and preservation. Using US, fully reproducible food processes can be accomplished in seconds or minutes with increased reliability, minimal processing cost, streamlined manipulation, elevated clarity to the end product, and expending only a fragment of the time and energy commonly required by conventional processes.This review emphasizes on the applications of ultrasound in different food sectors along with its certain limitations. Several operations such as microbial inactivation, enzyme inactivation, extraction, emulsification and fractionation in dairy industries, thermo-sonication in fruit juices have been discussed in detail. The US extracted dietary fiber consisted of increased amount of dietary fiber and trace elements in comparison to alkaline method. US initiate rapid creaming of milk fat, decreasing flavor loss and energy requirements thus enhancing the quality of end product. SWOT analysis has been carried out to pinpoint the strengths, weaknesses, opportunities and threats of sonication in various food industries.

Impact of pulsed light treatment on enzyme inactivation and quality attributes of whole white button mushroom (Agaricus bisporus) and its storage study

Whole white button mushrooms (WWBM) exhibit a limited shelf-life owing to the oxidative enzymatic browning. Inactivation of polyphenol oxidase-PPO and peroxidase-POD in WWBM and its kinetic behavior were studied using pulsed light(PL) treatment (0.13–1.11 J/cm2). The first-order kinetics explained PL-induced enzyme inactivation. Rate constants(k) for PPO and POD were 3.84 and 2.55 cm2/J. FTIR-analysis revealed secondary-structural changes in partially-purified enzyme. PL-treatment retarded browning, retained phenolics and enhanced vitamin D2. PL-treatment at 1.11 J/cm2 rendered WWBM both microbially and enzymatically stable. The PL-treated WWBM's shelf-life at 4, 20, and 37 °C were 5, 3, and 1 day. At 4 °C, browning increased by 6.1 %; firmness decreased by 55.2 %, while PL-treated mushrooms retained 90.6 % phenolics, 78.9 % antioxidant capacity, and 64.2 % D2 after 5 days. Higher activation energy value confirmed phenolics were most sensitive during storage. PL-technology supports UN Sustainable Development Goals by reducing chemical use, lowering carbon-footprints, minimizing pollution, and enhancing shelf-life, promoting sustainable global trade.

Changes in ficin specificity by different substrate proteins promoted by enzyme immobilization

Ficin extract has been immobilized using different supports: glyoxyl and Aspartic/1,6 hexamethylenediamine (Asp/HA) agarose beads. The latter was later submitted to glutaraldehyde modification to get covalent immobilization. The activities of these 3 kinds of biocatalysts were compared utilizing 4 different substrates, casein, hemoglobin and bovine serum albumin and benzoyl-arginine-p-nitroanilide at pH 7 and 5. Using glyoxyl-agarose, the effect of enzyme-support reaction time on the activity versus the four substrates at both pH values was studied. Reaction time has been shown to distort the enzyme due to an increase in the number of covalent support-enzyme bonds. Surprisingly, for all the substrates and conditions the prolongation of the enzyme-support reaction did not imply a decrease in enzyme activity. Using the Asp/HA supports (with different amount of HA) differences in the effect on enzyme activity versus the different substrates are much more significant, while with some substrates the immobilization produced a decrease in enzyme activity, with in other cases the activity increased. These different effects are even increased after glutaraldehyde treatment. That way, the conformational changes induced by the biocatalyst immobilization or the chemical modification fully altered the enzyme protein specificity. This may also have some implications when following enzyme inactivation.

Antioxidant and Cytoprotective Effects of Different Compounds on Cyclophosphamide-induced Nephrotoxicity

Abstract: Cyclophosphamide is a drug derived from nitrogenous mustards and is mainly indicated for the anti-neoplastic clinic. When it is metabolized in the liver, it produces acrolein, a cytotoxic metabolite capable of generating damage to the body. The oxidative, nitrative and nitrosative stress are responsible for mediating this side effect since the toxicity of the drug induces the production of reactive species of oxygen (ROS) and nitrogen (RNS). These radicals are unstable and react with other molecules that can result in cellular lesions such as lipid peroxidation, enzymatic inactivation, and excessive activation of pro-inflammatory genes, producing molecules such as malonaldehyde (MDA) and nitric oxide (NO), representing oxidative stress markers. One of the consequences of these mechanisms is nephrotoxicity. In contrast, some compounds have direct or indirect antioxidante action, which are the objects of this study: gallic acid, aminoguanidine, chrysin, hesperidine, naringin, morin-5'-sulfonic acid sodium salt and spirulina. All of them are responsible for the nephroprotective activity when administered during experiments in vitro or in vivo. Thus, this observational bibliographic research is characterized as qualitative descriptive and explanatory, since it focuses on analyzing the antioxidant properties of compounds with possible potentials to inhibit the nephrotoxicity induced by cyclophosphamide and describe the results, in addition to clarifying the biochemical activities involved in the process.

Dysfunctional High-Density Lipoprotein Cholesterol and Coronary Artery Disease: A Narrative Review.

High-density lipoprotein (HDL) cholesterol is traditionally viewed as protective against cardiovascular disease (CVD). However, emerging evidence reveals that dysfunctional HDL, characterized by impaired reverse cholesterol transport (RCT), reduced anti-inflammatory and antioxidant activities and increased endothelial dysfunction, which can contribute to coronary artery disease (CAD). Dysfunctional HDL, resulting from oxidative modifications of Apolipoprotein A-1 (Apo A-1) and enzyme inactivation, fails to effectively remove cholesterol from peripheral tissues and may promote inflammation and atherosclerosis. Genetic mutations affecting HDL metabolism further complicate its role in cardiovascular health. Studies have shown that conventional therapies aimed at raising HDL-C levels do not necessarily reduce cardiovascular events, highlighting the need for new approaches that improve HDL functionality. Therapeutic strategies such as Apo A-1 mimetic peptides, reconstituted HDL infusions, and drugs targeting specific HDL metabolic pathways are being explored. Additionally, weight loss, statin therapy, and niacin have shown potential in enhancing HDL function. The pathophysiology of dysfunctional HDL involves complex mechanisms, including oxidative stress, inflammation, and genetic mutations, which alter its structure and function, diminishing its cardioprotective effects. New functional assays, such as the cholesterol efflux capacity (CEC) and HDL inflammatory index, provide more accurate predictions of cardiovascular risk by assessing HDL quality rather than quantity. As research progresses, the focus is shifting towards therapeutic strategies that enhance HDL function and address the root causes of its dysfunction, offering a more effective approach to reducing cardiovascular risk and preventing CAD.