The effects of human wild-type and FALS mutant L144P SOD1 on non-vascular smooth muscle contractions

Background: Mutated copper, zinc-containing superoxide dismutase (SOD1) may self-aggregate, an event that could also be an initial cause of motor neuron malfunction leading to disease onset. The effects of human mutated SOD1 protein from the blood of familial amyotrophic lateral sclerosis (FALS) patients bearing Leu144Phe (L144F) mutation were compared to wild-type (WT) human SOD1 derived from healthy examinees, for enzymatic activity and the effects on isometric contractions of non-vascular smooth muscle. Methods: We isolated WT and L144F SOD1 enzymes from eight patients with FALS, L144F mutation in exon 5 and eight healthy controls. We then investigated SOD1 activities in the obtained samples by the adrenaline method and profiled them electrophoretically. Finally, we applied WT and L144F SOD1 on the isolated rat uterus. Results: L144F SOD1 showed lower superoxide-dismutating activity compared to WT human SOD1. We found that, in contrast to WT human SOD1, mutated L144F does not induce smooth muscle relaxation. Conclusions: Our data suggest that the lack of relaxation of muscle tonus in the presence of mutated SOD1 may have pathogenic feedback effects in FALS.Uvod: Mutirana bakar, cink superoksid-dizmutaza (SOD1) može da pravi agregate, sto predstavlja početni uzrok oštećenja motornog neurona može da izazove nastanak bolesti. U ovom radu su pokazani efekti humane bakar, cink super-oksid dizmutaze iz krvi pacijenata obolelih od familijarne amiotrofične lateralne skleroze (FALS) sa Leu144Phe (L144F) mutacijom i normalne (wild-type - WT) humane SOD1, iz krvi zdravih kontrola, na glatkom mišiću. Metode: Izolovali smo WT i L144F SOD1 enzime kod osam odabranih FALS pacijenata sa L144F mutacijom na egzonu 5 i pet zdravih kontrola. Dalje smo ispitivali aktivnost SOD1 u dobijenim uzorcima adrenalinskom metodom i elektro-foretski ih profilisali. Konačno, izolovanu WT i L144F SOD1 aplicirali smo na izolovani uterus pacova. Rezultati: Aktivnost L144F SOD1 je statistički značajno manja (p<0,05) u poređenju sa aktivnosti WT SOD1 zdravih kontrola. L144F ne izaziva relaksaciju glatkog mišića, kao sto je to slučaj sa WT SOD1. Zaključak: Naši rezultati pokazuju da izostanak relaksacije mišićnog tonusa u prisustvu mutirane SOD1 može imati štetni povratni efekat kod FALS pacijenata.Projekat ministarstva br. 173014 i br. 17508

The effects of human wild-type and FALS mutant L144P SOD1 on non-vascular smooth muscle contractions

Uvod: Mutirana bakar, cink superoksid-dizmutaza (SOD1) može da pravi agregate, sto predstavlja početni uzrok oštećenja motornog neurona može da izazove nastanak bolesti. U ovom radu su pokazani efekti humane bakar, cink super-oksid dizmutaze iz krvi pacijenata obolelih od familijarne amiotrofične lateralne skleroze (FALS) sa Leu144Phe (L144F) mutacijom i normalne (wild-type - WT) humane SOD1, iz krvi zdravih kontrola, na glatkom mišiću. Metode: Izolovali smo WT i L144F SOD1 enzime kod osam odabranih FALS pacijenata sa L144F mutacijom na egzonu 5 i pet zdravih kontrola. Dalje smo ispitivali aktivnost SOD1 u dobijenim uzorcima adrenalinskom metodom i elektro-foretski ih profilisali. Konačno, izolovanu WT i L144F SOD1 aplicirali smo na izolovani uterus pacova. Rezultati: Aktivnost L144F SOD1 je statistički značajno manja (p lt 0,05) u poređenju sa aktivnosti WT SOD1 zdravih kontrola. L144F ne izaziva relaksaciju glatkog mišića, kao sto je to slučaj sa WT SOD1. Zaključak: Naši rezultati pokazuju da izostanak relaksacije mišićnog tonusa u prisustvu mutirane SOD1 može imati štetni povratni efekat kod FALS pacijenata.Background: Mutated copper, zinc-containing superoxide dismutase (SOD1) may self-aggregate, an event that could also be an initial cause of motor neuron malfunction leading to disease onset. The effects of human mutated SOD1 protein from the blood of familial amyotrophic lateral sclerosis (FALS) patients bearing Leu144Phe (L144F) mutation were compared to wild-type (WT) human SOD1 derived from healthy examinees, for enzymatic activity and the effects on isometric contractions of non-vascular smooth muscle. Methods: We isolated WT and L144F SOD1 enzymes from eight patients with FALS, L144F mutation in exon 5 and eight healthy controls. We then investigated SOD1 activities in the obtained samples by the adrenaline method and profiled them electrophoretically. Finally, we applied WT and L144F SOD1 on the isolated rat uterus. Results: L144F SOD1 showed lower superoxide-dismutating activity compared to WT human SOD1. We found that, in contrast to WT human SOD1, mutated L144F does not induce smooth muscle relaxation. Conclusions: Our data suggest that the lack of relaxation of muscle tonus in the presence of mutated SOD1 may have pathogenic feedback effects in FALS

A search for nature’s robust proteases with zein as a substrate

Zein is produced in large quantities as a byproduct of corn starch manufacturing since it constitutes a majority of the total protein of maize seed (44–70%). Enzymatic treatment of zein significantly improves its aqueous solubility and provides peptides that are used as animal feed, functional food, or biologically active carriers for other bioactive molecules. Moreover, zein-derived peptides exhibit antioxidant, anti-inflammatory, antihypertensive, anticancer, and antimicrobial activities in human organisms 1. Few attempts up to this day have been made to screen for microorganisms that are capable of zein degradation. Available protocols for proteases identification almost exclusively rely on screening on casein, skim milk, and gelatin agar in limited experimental conditions. We have screened different Bacillus sp strains isolated from across Serbia for zein-degrading proteases. To do so we developed an inexpensive, simple, and reproducible way of high throughput functional screening of zein-degrading proteases on zein-containing gels. Besides detecting proteases with specificity towards zein, a developed diffusion assay was designed to support screening for naturally occurring robust proteases with high potential for industrial application. By using classical methods of protein purification, we isolated an alkaline thermostable protease from Bacillus amyloliquefaciens strain 12B that is resistant to the presence of detergents, organic solvents, and high salt concentrations

The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs) are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC) treatment on superoxide-dismutase (SOD) activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH)24) at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET). The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL) after 3 and 24 h of cell incubation. Increasing of C60(OH)24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL) + MMC was noticed boost in total SOD activity in comparison with controls. Treatment with fullerenol decreased SOD activity in rest of samples treated with MMC. Decreased activity of superoxide-dismutase in almost all samples treated with fullerenol and MMC might be contributed to antioxidative properties of fullerenol. Increased enzyme level at concentration of 0.0625 mg/mL may be due to its prooxidative activity.U ovom radu ispitivani su efekti fulerenola (C60(OH)24) na preživljavanje, kao i tretmana fulerenolom i mitomicinom c (MMC) na aktivnost ukupne superoksid-dismutaze u CHO-K1 (ovarijalnih ćelija hrčka) ćelijskoj liniji. U uzorcima ćelija tretiranim fulerenolom koncentracija 0,01-0,5 mg/mL, praćeno je preživljavanje testom odbacivanja boje (DET) u 3 i 24 h tretmanu. Aktivnost superoksid-dismutaze (SOD) merena je u uzorcima tretiranim fulerenolom izabranih koncentracija i mitomicinom c (0,5 i 0,1 mg/mL) nakon 3 i 24 h. Sa porastom koncentracije (C60(OH)24) opada procenat preživelih ćelija tokom 3 i 24 h. Aktivnost SOD raste sa porastom koncentracije fulerenola i u najvećoj koncentraciji opada u obe vremenske tačke eksperimenta. U uzorcima tretiranim fulerenolom i MMC došlo je do smanjenja aktivnosti SOD, izuzev pri koncentraciji fulerenola od 0,0625 mg/mL, kada je zapažen porast aktivnosti SOD u odnosu na kontrolne grupe.Projekat ministarstva br. 14207

Screening of pectinase-producing Aspergillus spp. for use in strawberry juice clarification

U ovom radu urađen je skrining sojeva Aspergillus spp. koji proizvode pektinaze fermentacijom u tečnom medijumu, a koje su efikasne u izbistravanju soka od jagode. Endo-pektinaze su detektovane difuzionim testom, a ukupna pektinazna aktivnost određena je DNS metodom. Izbistravanje soka nakon tretmana pektinazama određeno je merenjem transmitancije na 660 nm, a povećanje prinosa soka izmereno je centrifugiranjem. Dobijeni rezultati su pokazali da su ove pektinaze visokoefikasne za tretman soka od jagode u poređenju sa komercijalnim pektinaznim preparatom Lafase Fruit.In this work, the screening of Aspergillus spp which produce pectinases by fermentation in a liquid medium was performed, as pectinases are effective in clarifying strawberry juice. Endo-pectinases were detected by the diffusion assay, and total pectinase activity was determined by the DNS method. The clarification of juice after pectinase treatment was determined by measuring the transmission at 660 nm, and an increase in juice yield was measured by centrifugation. The results obtained showed that these pectinases are very effective for the treatment of strawberry juice in comparison with the commercial pectinase preparation Lafase Fruit.59th Meeting of the Serbian Chemical Society; June 1-2, 2023, Novi Sad, Serbi

Cloning, overexpression and characterization of a thermostable endo-1,4-beta-xylanase from Anoxybacillus vranjensis ST4

This research deals with the characterization of a thermostable endo-1,4-beta-xylanase enzyme from Anoxybacillus vranjensis ST4, a thermophilic bacterium isolated from Vranjska Banja hot spring, Serbia. The enzyme shows a high degree of identity with the same type of enzyme from other species of the genera Anoxybacillus (97%), Geobacillus (74%) and Paenibacillus (65%). The gene for endo-1,4-beta-xylanase from the thermophilic strain ST4 was cloned into the pQE_Ek expression vector and successfully expressed and purified from the Escherichia coli M15[pREP4]. The study encompasses recombinant production, purification, and the comprehensive characterization of the enzymatic properties of endo-1,4-beta-xylanase. This is the first successful overexpression, purification and characterization of a recombinant thermostable endo-1,4-beta-xylanase enzyme from Anoxybacillus. With a monomeric structure of 38.7 kDa, the enzyme demonstrates peak activity at 70°C and pH 6.5. Notably, it exhibits remarkable stability across a wide pH range and at high temperatures, rendering it suitable for diverse industrial applications. Investigation into the enzyme’s kinetic parameters, substrate specificity, and its ability to degrade xylan into high-energy value products further enhances understanding of its biotechnological potential. These findings underscore the significance of thermophilic bacteria and their thermostable enzymes in various industrial processes

Occupational diseases and general practitioners

Cilj ovog istraživanja je istražiti mišljenja liječnika obiteljske medicine o prijavljivanju profesionalnih bolesti, mogućoj povezanosti radnog statusa, bolovanja i ocjene radne sposobnosti s profesionalnom bolesti njihovih pacijenata, o potrebi poboljšanja suradnje sa specijalistima medicine rada i potrebi liječnika obiteljske medicine za edukacijom u pojedinim područjima zdravlja na radu i profesionalnih bolesti te temama i načinima provedbe edukacije. Ispitanici su bili liječnici, specijalisti obiteljske medicine i liječnici opće medicine koji su zaposleni u ambulantama obiteljske medicine u Zagrebu. Analizirane su prijavljene profesionalne bolesti u ambulantama obiteljske medicine. Od ukupnog broja liječnika obiteljske medicine (N=50), 26 % prijavilo je profesionalnu bolest kod svojih osiguranika, 70 % nije prijavilo, a njih 4 % nisu znali jesu li u posljednjih 5 godina prijavili profesionalnu bolest. U skupini prijavljenih profesionalnih bolesti (N=13) najzastupljenije su bile bolesti mišićno-koštanog sustava (38 %) i kožne bolesti (38 %). Najčešći razlozi neprijavljivanja bili su nepoznavanje kauzaliteta i sprege s radnim mjestom kod 91 % liječnika, te u 9 % slučajeva i strah da će pacijent dobiti otkaz. Velika većina ispitanika (94 %) slaže se da je potrebno poboljšati suradnju između obiteljskih liječnika i specijalista medicine rada. Zainteresiranost za edukaciju iskazalo je 78 % obiteljskih liječnika, a potreba postoji za edukacijom iz područja prepoznavanja profesionalnih bolesti. Timskim preventivnim radom specijalista medicine rada i sporta i obiteljskih liječnika, uporabom „check lista“ u ordinacijama obiteljske medicine i odabranim edukativnim tečajevima iz područja zdravlja na radu unaprijedila bi se primarna prevencija profesionalnih bolesti, a ranim otkrivanjem spriječila daljnja oštećenja zdravlja i smanjila invalidnost koja nastaje kao posljedica obolijevanja od profesionalnih bolesti.The aim of this study is to investigate the views of general practitioners regarding the reporting of occupational diseases and the possible link between work status, sickness and assessment of work ability with occupational diseases. The study also places focus on the need to improve collaboration with occupational medicine specialists, on general practitioners\u27 need for education in certain areas of occupational health and occupational diseases, and on topics and ways of implementing education. The subjects were family physicians and general practitioners (50) employed in community health centers in Zagreb. The reported occupational diseases in family physician\u27s surgeries were analyzed. It was found that 26% respondents reported occupational disease, 70% did not report it, and 4% did not know whether or not they had reported occupational disease in the last five years. The most commonly reported occupational diseases were diseases of the musculoskeletal system (38%) and skin diseases (38%). The reasons given for not reporting occupational diseases were that doctors were not aware that those were occupational diseases (91%), and because they feared that the patient may get fired (9%). The vast majority of respondents (94%) agreed that it is necessary to improve cooperation between family physicians and occupational medicine specialists. Seventy eight percent of family physicians were interested in education on how to recognize occupational diseases. Preventive team work on the part of occupational medicine specialists and family physicians using the \u27check lists\u27 in family physician\u27s surgeries, and education courses on occupational health would improve primary prevention of occupational diseases, while early detection would halt the progress of occupational diseases and thus reduce ensuing disability caused by occupational disease

Highly active endo-pectinases from Aspergillus tubingensis: A novel enzyme for fruit processing

Pectinases are a type of enzymes frequently used in the food industry to clarify, liquefy, and stabilize fruit juices. The main challenge in fruit juice production is the cloudiness of the juice, which is largely caused by the presence of pectic polysaccharides. Endo-pectinases are enzymes that hydrolyze the glycosidic bonds in pectic polymers. Commercial pectinolytic enzymes are typically produced by fungi, with Aspergillus spp. being the most commonly used. The aim of this research was the production and characterization of a novel endo-pectinase from the Aspergillus tubingensis strain for use in liquefying and clarifying different types of fruit juice. To accomplish this, solid-state fermentation was conducted on agricultural waste, such as sugar beet pulp and wheat bran, to produce pectinolytic enzymes. The resulting crude extract was concentrated via ultrafiltration and used to isolate the endo-pectinase via ammonium sulfate and ethanol precipitation methods. Ion-exchange chromatography technique on DEAE Sephadex A-25 matrix was used for further purification of the endo-pectinase. The purified enzyme was characterized by the determination of total pectinolytic activity, specific pectinolytic activity, and SDS-PAA gel electrophoresis. The activity of the endo-pectinase was confirmed by a diffusion test and zymography with Ruthenium Red visualization. The resulting enzyme was used to liquefy apricot, banana, apple, quince, strawberry, and orange pulp, with juice yields ranging from 71% to 83%, depending on the fruit used. The juices treated with endo-pectinase showed much higher clarification compared to untreated juices. Additionally, the treated juices demonstrated more pronounced antioxidant properties, as determined through the DPPH assay

Exploring the biotechnological potential of thermophilic bacteria - derived pectin lyases: a mini-review

Bacteria are an ideal source for producing pectin lyases (PNLs) due to their amenability to laboratory cultivation and genetic manipulation, which facilitates enhanced enzyme production. Predominantly originating from various thermophilic bacteria, bacterial PNLs usually exhibit alkaline properties, although cases of acidic variants have also been documented. In particular, a thermostable alkaline pectin lyase, displaying optimal activity at 60°C, has been characterized from the thermophilic bacterium Brevibacillus borstelensis P35. Similarly, thermostable acidic PNLs have been identified in Geobacillus stearothermophilus Ah22 and Bacillus subtilis SAV-21. Thermophilic bacterial species are emerging as significant and highly efficient sources, boasting diverse enzymatic repertoires, including pectinolytic enzymes, rendering them attractive candidates for various biotechnological applications. This mini-review focuses on the characterization of pectin lyases from a thermophilic bacterium, shedding light on its biochemical properties, substrate specificity, and potential industrial applications. Enzymes exhibit outstanding biochemical properties, with optimal pH and temperature ranges conducive to industrial processes, along with notable thermostability and pH tolerance, augmenting their suitability for diverse biotechnological endeavours. Furthermore, the enzyme demonstrates specificity towards pectin, efficiently cleaving glycosidic bonds within the polysaccharide backbone. Understanding the substrate specificity of pectin lyases is crucial for its effective utilization in industrial processes, especially considering its preferences for high-methoxylated pectin while still demonstrating activity on low-methoxylated and amidated pectins, expanding its applicability. Additionally, the synergy of pectin lyases with other pectinolytic enzymes enhances the efficiency of pectin degradation, facilitating the production of valuable products such as biofuels, dietary fibers, and oligosaccharides. The versatility and efficiency of pectin lyases from thermophilic bacteria highlight its potential for application across various biotechnological sectors, including food and beverage, textile, and pharmaceutical industries. Its capability to modify pectinaceous materials offers sustainable solutions for waste valorization and bioconversion processes

Expression of protease in adult honey bees fed with different patties

Feeding of honey bee (Apis mellifera) is a challenge for beekeepers and formulation of food supplements is improved continuously. When natural food sources are scarce or not available, supplemental foods are widely used to support and build up honey bee colonies. Influence of commercial (sugar) patty and the patties enriched with 12.5% pollen and 12.5% dried yeast on protease expression in honey bee adults is presented. This is part of a wider study aimed to compare the expression of digestive enzymes using different patties. Data collected in this study can be useful for development of higher quality of food supplements for honey bees. Honey bees were kept in an incubator for 21 days, at a temperature of 35°C and at 80% humidity. In each cage there were one hundred bees and a piece of honeycomb. Midgut samples were taken after 7, 14 and 21 days, homogenized and analyzed for protein concentration and protease activity. The highest protein concentration was detected in the midgut of pollen fed bees, and lowest in commercial patty fed group, determined by the Bradford method. Protease activity was the highest in bee’s midgut fed with pollen patties, and the lowest in bees fed commercial patties which is shown by enzyme assay and by zymograms. There were different protease isoforms present in bees fed pollen, yeast and commercial patties, but the major isoforms were the same. The observed decrease in protease activity over time in all groups is probably due to the transition to foraging tasks. Reduced protease activity in the gut of bees fed commercial patties is due to a lack of proteins or some other inducers present in food. Because pollen is honey bee natural food, we conclude that food supplements that induce similar enzyme expression as pollen (dried yeast) can be considered as appropriate food substitution, because it is superior to supplements which induce very different enzyme expression.Abstract book: [https://doi.org/10.5281/zenodo.7239012