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The aim of this study was to investigate the morphological traits and fragrance-related volatile compounds in three garden rose cultivars from the 'Fragrant Frayla' collection: 'Draga Frayla', 'Jelena Frayla', and 'Lenka Frayla', bred by the private breeding company Pheno Geno Roses. The roses were cultivated under open-field conditions in Temerin, Serbia, without irrigation and chemical treatments. Vegetative and generative properties were assessed in June and September 2023, while headspace volatile extraction and gas chromatography–mass spectrometry (GC-MS) were employed to identify scent-related volatiles. All three cultivars exhibited high ornamental value, with shrub heights ranging from 47 to 67 cm, semi-upright to upright growth habits, double flowers with diameters from 6.6 to 8.2 cm, and petal counts from 35.8 to 83.8. Upon sensory evaluations, 'Draga Frayla', 'Jelena Frayla', and 'Lenka Frayla' were characterized by moderately strong, strong and moderate fragrances, respectively. Volatile analysis detected the presence of phenylethyl alcohol (up to 57.31% in 'Draga Frayla'), terpenoids (a total of 28.2% for citronellol, geraniol, and nerol, found exclusively in 'Jelena Frayla'), and hydrocarbons. Hydrocarbons containing fewer than 30 C-atoms were predominantly found in 'Draga Frayla' (accounting for approximately 25% of the total peak area), while those with more than 30 C-atoms were present at less than 20% in the strongly scented roses. The highest number of volatiles was detected in 'Jelena Frayla', the rose cultivar identified as the most fragrant. The results revealed a correlation between strong fragrance intensity and elevated levels of phenylethyl alcohol and alcoholic monoterpenes, whereas a diverse array of hydrocarbons, rather than their concentration, appeared to enhance scent emission.

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Ferritin is the main intracellular storage of iron. Animal studies show that female liver and kidney express more ferritin and accumulate more iron than male. However, no study so far has investigated sex and age differences in light (FtL) and heavy (FtH) ferritin chain expression. To address this, we relied on specific antibodies and immunochemical methods to analyse the expression of both ferritin chains in the liver and kidney of 3-month and 2-year-old male and female Wistar rats. To see how sex hormones may affect expression we also studied adult animals gonadectomised at the age of 10 weeks. FtL and FtH were more expressed in both organs of female rats, while gonadectomy increased the expression in males and decreased it in females, which suggests that it is stimulated by female and inhibited by male steroid hormones. Normal kidney ferritin distribution and change with aging warrant more attention in studies of (patho) physiological and toxicological processes.

Severe climate alterations that seriously challenge fruit production, combined with the demand for healthy, pesticide-free fruits, continuously direct rootstock/cultivar selection towards high adaptable varieties breeding. This study aimed to investigate the rootstocks’ influence on the performance of grafted ‘Summit’ cherry trees, including potentially dwarfing Prunus cerasus, Prunus fruticosa and Prunus mahaleb rootstock candidates. Anatomical properties of rootstock and scion stems were investigated to determine variation among different rootstocks and scion-rootstock combinations and to establish the link between trunk hydraulic conductivity, effective tree crown volume and yielding potential. Cross-section anatomical characteristics varied significantly both in rootstock and scion stems, indicating a clear influence of rootstock genotype on grafted sweet cherry trees. It was observed that all investigated cherry rootstock candidates belong to the low-vigorous rootstocks, based on the estimated effective crown volume of grafted trees compared to ‘Gisela 5’, with values ranging from 0.86 to 2.97 m3 in the fifth year after planting. Results showed a statistically significant positive correlation between trunk hydraulic conductivity, effective tree crown volume and yielding potential, with correlation coefficients up to 0.96. Significantly higher effective crown volume and trunk hydraulic conductance of trees grafted on P. cerasus compared to the trees on control, as well as highest yielding potential, showed better adaptation of these rootstock candidates in the trial without irrigation implemented. It was found that PC_05_04 rootstock candidate could be considered as the most appropriate choice when raising the high-density sweet cherry plantations, due to assessed parameters of vegetative and generative growth.

Temperature increase in urban areas due to a high share of paved and impermeable surfaces affects indigenous woody species by making changes in their biological processes. Field research includes monitoring the blooming period of 18 individuals of Tilia tomentosa Moench. in one vegetation season located in three different habitats (green strip, paved surface, and park). The beginnings of four blooming phenophases are monitored (beginning of blooming, beginning of full blooming, end of full blooming, and end of blooming), as well as the total duration of blooming. Analyzed individuals are located in the densely built-up part of Novi Sad in Serbia which is under the influence of the urban heat island effect. The results show that the blooming period of individuals located in the park lasted longer and occurred later. Between individuals from green strips and paved surfaces there are no significant dif­ferences in the blooming period and the occurring of phases, but in relation to park individuals, blooming duration is shorter and occurs earlier. Besides age and hereditary traits, air temperature as a part of the microclimate of analyzed habitats is a very important factor when analyzing the blooming phenophase. In that way, the change in blooming phenophases can be an indicator of the adaptability of indigenous species to the urban heat effect and climate change.

Study aim: The body structure can play a determining role in the achievement of top judo performance, and it seems to influence the type of techniques applied. The aim of this study is to determine the somatotypes in male and female national level judokas across weight categories in order to observe possible differences among athletes.

Sweet cherries (Prunus avium L.) contain various phenolic compounds which contribute to total antioxidant activity. The present study was conducted to assess the antioxidant activity of 15 sweet cherry cultivars from Vojvodina province (north Serbia). The free radical scavenging properties of fruits were evaluated employing two different methodologies, including DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay and ferric reducing antioxidant power (FRAP) assay. Strong correlations were found between total phenolics, tannins, flavonoids and anthocyanins and DPPH and FRAP assays. In particular, cultivar Peter showed the highest antioxidant capacity and possesses the highest amount of measured phenolic compounds. This investigation shows large variability among sweet cherry cultivars in measured chemical attributes.

Ophrys sphegodes Mill. is a wild orchid species which is threatened and protected due to its pollination biology, small seed and habitat destruction. The aim of this study was to establish asymbiotic germination protocol for the purpose of ex situ conservation. Two basal media Knudson C (KC) and Malmgren (MM), supplemented with organic additives (peptone (PE), L-glutamin (A)e, folic acid, casein hydrolysate (CA)) added separately and control media KC--C and MM--C were used in the present research. All the nutrition media contained 2% sucrose, 7% agar and 1% activated carbon, while their pH was adjusted to 5.8 ± 0.02 before autoclaving at 121 ° C for 20 minutes. The seeds were examined under two illumination conditions, 0/24 light/dark (L/D) and 16/8 L/D. The presented results indicate a huge influence of illumination and nutrition media on the seed germination and protocorm formation. The seed germination was overall significantly more successful in dark conditions (0/24 L/D) than with lighting (16/8 L/D). Protocorm, rhizoids and shoot formation were achieved only on the seeds cultured on MM medium, while the KC medium caused only swelling of the embryo. Organic additives had positive influence on the germination rate. According to the obtained results, the best germination rate and seedling development were achieved on MM-PE media, cultured in dark. The presented procedure accelerates the germination period and can provide a large number of plants in a relatively short period of time so it can be used for conservation programs and mass production protocol.

The purpose of this paper is to analyze relations between seed quality characteristics and the influence of contaminated urban areas on seed quality of sycamore maple trees in different locations across Novi Sad (Serbia). This research analyzed seed germination of 29 adult trees of sycamore maple (Acer pseudoplatanus L.) and its red leaf variety (Acer pseudoplatanus ‘atropurpureum’ Späth.). Our results indicate a medium strong positive correlation between seed weight and viable seed. Sycamore maple trees from the same urban typology do not have significant influence on seed quality characteristics, while different urban typology has such influence. These results explain the negative influence of urban areas on seed quality characteristics. For selection of high value trees in urbanized and contaminated areas, seed quality can be the indicator of adaptability to stress related conditions.

This paper shows the results of the analysis of the fruit morphometric characteristics of 29 trees of sycamore maple (Acer pseudoplatanus L.) and red - leaf sycamore maple (Acer pseudoplatanus ‘Аtropurpureum’ Späth.) in Novi Sad area. Based on the test trees, it can be concluded that the analyzed secondary population of sycamore maple has a high level of intra - populations variability, based on different degrees of variability of measured parameters and statistically significant differences of all analyzed parameters within the analysed genotypes. The results indicate that there are certain differences between fruit of sycamore maple and its red - leaf variety. Given that there is no statistically significant difference between sites and different urban spaces, it can be concluded that stress factors caused by a high degree of urbanity do not affect the morphometric characteristics of fruits in the analyzed test trees. Testing the symmetry of fruits indicates a high level of genetic variability within the analyzed population.

Variability and correlations among morphological features of eight ornamental pumpkin varieties were studied under field conditions. The variability of plant height, fruit length, fruit width, fruith weight, fruit peel thickness, length and circumference of handle grip, leaf length, leaf width, seed length, seed width, seed thickness and number of fruits per plant in the examined material was high. The highest variability was related to the fruit properties. This variability represents a good source for future breeding programs. Correlations between the traits indicated a significant influence of leaf and seed characteristics on fruit properties. Multivariate statistical analysis provided differentiation of varieties on two phenotypically different groups.

A wide range of bottle gourd Lagenaria siceraria (Mol.) Standley germplasm was collected from different parts of the world. Genetic resource preservation and determination of genetic variability was carried out as a foundation for future breeding work. The germplasm diversity collected in Serbia is a result of its adaptation to diverse ecological conditions and farmers’ selection in accordance with their preference and ethnobotanical utilization. The broad intraspecific variation of the plant, fruit and seed morphology is a direct result of the research carried out. Principal component analysis (PCA) of L. siceraria with 13 quantitative traits showed continuous variation among accessions, primarily due to fruit and seed size and shape. The evident reduction in trait variation is a direct result of the preference for ornamental use that favored certain shapes and sizes of the fruit, which has not significantly changed over the centuries.

This study presents research on the chemical composition of 15 horse chestnut seed genotypes (Aesculus hippocastanum L.) conducted on three different sites. The seeds were picked from selected trees in Bačka Palanka, Novi Sad and Sremski Karlovci at the end of September 2010. The statistical parameters point out the variables of the examined features of the seed. On the basis of the moisture, starch, fat and protein contents in the horse chestnut seeds, the genotypes, the reproductive material which would be used for the intensive production of the high grade seedlings for the greening of the different area categories in urban areas, are distinguished.

Although they have been extensively studied in many species of the genus Rosa L., garden roses' hips have largely been overlooked. To investigate their potential use in the food industry, this study evaluated five cultivars from 'Aurora' collection: 'Purple Aurora', 'Berry Bush Aurora', 'Aromatic Aurora', 'Butterfly Aurora', and 'Rugose White Aurora'. Morphological characterization, along with the assessment of the phenolic profile, vitamin C levels, and biological activities-including antioxidant and neuroprotective effects-was conducted. The fruit mass reached 5.15 g, while the mesocarp mass ranged from 3 to 4 g, resulting in a mesocarp-to-fruit ratio of over 75%. The total phenolic content ranged from 37.1 to 63.9 mg GAE/g de, while total flavonoids were present in amounts from 0.85 to 2.14 mg QE/g de. Rosehip extracts from four out of five cultivars exhibited a very high vitamin C content, reaching 2384 µg/g fw. Quinic acid and 31 phenolic compounds were found in the rosehip extract of at least one cultivar. Results indicated 'Aurora' rosehips have potent antioxidant properties and a moderate inhibitory effect on acetylcholinesterase, highlighting their potential as a source of functional food. Additional research is needed to fully leverage these benefits and establish garden rosehips as a viable alternative to synthetic antioxidants.

Garden roses, known as Rosa × hybrida , hold a prominent position as one of the most important and economically valuable plants in horticulture. Additionally, their products-essential oil, rose water, concrete, and concentrate-find extensive use in the cosmetic, pharmaceutical, and food industries, due to their specific fragrances and potential health benefits. Rose flowers are rich in biologically active compounds, such as phenolics, flavonoids, anthocyanins, and carotenoids. This study aims to investigate the potential of five new garden rose genotypes with intensely colored flowers to serve as sources of biologically active compounds. Phenolic profile was evaluated by determination of total phenolic (TPC), flavonoid (TFC), and monomeric anthocyanins (TAC) contents and LC-MS/MS analysis of selected compounds. Antioxidant activity was evaluated via DPPH and FRAP assays, neuroprotective potential via acethylcholinesterase inhibition assay, and antidiabetic activity viaα-amylase and α-glucosidase inhibition assays. The flowers of investigated genotypes were rich in phenolics (TPC varied from 148 to 260 mg galic acid eq/g de, TFC from 19.9 to 59.7 mg quercetin eq/g de, and TAC from 2.21 to 13.1 mg cyanidin 3- O -glucoside eq/g de). Four out of five genotypes had higher TPC than extract of R. damascene , the most famous rose cultivar. The dominant flavonoids in all investigated genotypes were glycosides of quercetin and kaempferol. The extracts showed high antioxidant activity comparable to synthetic antioxidant BHT, very high α-glucosidase inhibitory potential, moderate neuroprotective activity, and low potential to inhibit α-amylase.

The green infrastructure of the city of Novi Sad is characterized by a significant presence of ornamental invasive alien species, which might lead to their uncontrolled spread and suppression of autochthonous dendroflora. This study aimed to determine the ecosystem services and disservices of ornamental dendroflora in Novi Sad, as well as how they can affect urban green areas. Of the total ornamental dendroflora in Novi Sad, 88.33% of species with a good adaptation were determined, while 10% had a medium and 1.67% had a very good adaptation. Thirty-four allochthonous species showed very high (38.24%), moderate (47.06%), and low (14.71%) invasive potential according to the conducted invasiveness risk assessment. These species are also characterized by high (2.94%), moderate (67.65%), and low (29.41%) allergenic potential. On the contrary, 26 ornamental autochthonous species are characterized by moderate (38.46%) and low (61.54%) rates of spread on public green areas, while also characterized by high (26.92%), moderate (50%), and weak (23.08%) allergenic potential. Ornamental dendroflora provides many more positive ecosystem services, such as urban afforestation, climate regulation, decorative-aesthetic value, air and water purification, ecotourism and recreation, and other services that are of great benefit to the residents of that city. The highest calculated values of ecosystem services in allochthonous and autochthonous species were 27 and 26.5 (out of possible maximal value 40), while ecosystem disservices accounted down to the value of - 13.5 (out of possible minimal value - 22). Therefore, intrinsic disservices such as the production of large amounts of green waste of ornamental dendroflora can be shifted into a novel ecosystem service-green solutions based on nature, to avoid unsuitable deposition of seeds in the soil and creation of suitable vegetation on public green areas., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aging-related impaired body structure and functions may be, at least partially, caused by elevated oxidative stress. Melatonin (MEL) and resveratrol (RSV) may act as antioxidant and anti-aging compounds, but these actions in experimental animals and humans are controversial. Herein, a rat model of aging was used to study the long-term sex-related effects of MEL and RSV treatment on body mass and blood/plasma parameters of DNA damage, oxidative status (glutathione and malondialdehyde levels), and concentrations of sex hormones. Starting from the age of 3mo, for the next 9mo or 21mo male and female Wistar rats (n = 4-7 per group) were given water to drink (controls) or 0.1 % ethanol in water (vehicle), or MEL or RSV (each 10 mg/L vehicle). DNA damage in whole blood cells was tested by comet assay, whereas in plasma, glutathione, malondialdehyde, and sex hormones were determined by established methods. Using statistical analysis of data by ANOVA/Scheffe post hoc, we observed a similar sex- and aging-dependent rise of body mass in both sexes and drop of plasma testosterone in control and vehicle-treated male rats, whose pattern remained unaffected by MEL and RSV treatment. Compared with controls, all other parameters remained largely unchanged in aging and differently treated male and female rats. We concluded that the sex- and aging-related pattern of growth and various blood parameters in rats were not affected by the long-term treatment with MEL and RSV at the estimated daily doses (300-400 μg/kg b.m.) that exceed usual moderate consumption in humans., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Silver nanoparticles (AgNPs) are widely used in biomedicine due to their strong antimicrobial, antifungal, and antiviral activities. Concerns about their possible negative impacts on human and environmental health directed many researchers towards the assessment of the safety and toxicity of AgNPs in both in vitro and in vivo settings. A growing body of scientific information confirms that the biodistribution of AgNPs and their toxic effects vary depending on the particle size, coating, and dose as well as on the route of administration and duration of exposure. This study aimed to clarify the sex-related differences in the outcomes of oral 28 days repeated dose exposure to AgNPs., Methods: Wistar rats of both sexes were gavaged daily using low doses (0.1 and 1 mg Ag/kg b.w.) of polyvinylpyrrolidone (PVP)-coated small-sized (10 nm) AgNPs. After exposure, blood and organs of all rats were analysed through biodistribution and accumulation of Ag, whereas the state of the liver and kidneys was evaluated by the levels of reactive oxygen species (ROS) and glutathione (GSH), catalase (CAT) activity, superoxide dismutase (SOD) and glutathione peroxidase (GPx), expression of metallothionein (Mt) genes and levels of Mt proteins., Results: In all animals, changes in oxidative stress markers and blood parameters were observed indicating the toxicity of AgNPs applied orally even at low doses. Sex-related differences were noticed in all assessed parameters. While female rats eliminated AgNPs from the liver and kidneys more efficiently than males when treated with low doses, the opposite was observed for animals treated with higher doses of AgNPs. Female Wistar rats exposed to 1 mg PVP-coated AgNPs/kg b.w. accumulated two to three times more silver in the blood, liver, kidney and hearth than males, while the accumulation in most organs of digestive tract was more than ten times higher compared to males. Oxidative stress responses in the organs of males, except the liver of males treated with high doses, were less intense than in the organs of females. However, both Mt genes and Mt protein expression were significantly reduced after treatment in the liver and kidneys of males, while they remained unchanged in females., Conclusions: Observed toxicity effects of AgNPs in Wistar rats revealed sex-related differences in response to an oral 28 days repeated exposure., (© 2021. The Author(s).)

Due to Koelreuteria paniculata Laxm., high abundance in Novi Sad (Serbia) and previously confirmed biodiesel feedstock suitability, this study aimed to assess generative potential and yield, assess K. paniculata invasive risk, and investigate the influence of harvesting periods on the oil content and quality. Fifty-five specimens present in the Novi Sad inner-city core exhibited very high scores in the conducted invasiveness risk assessment (score 35/39). Determined good overall adaptability, growth and development, vitality and decorativeness of K. paniculata specimens, as well as the absence of phytopathological and entomological damages, due to lack of natural enemies, provided this species high unhindered yielding. The majority of investigated trees reached 5-10 m in height and canopy volumes from 10.1 to 70 m 3 , with an estimated more than 130 000-700 000 seeds produced per canopy. Seeds from one representative specimen were collected on different harvesting dates, at the end of August, September and October of 2019, and analyzed for oil content and quality for biodiesel production. With the average oil percentage of 22.8 w%, determined in the representative specimen, investigated 55 seed-bearing K. paniculata trees merely in the inner city core of Novi Sad, could produce 115 kg of oil. Since oil properties (<1 w% of free fatty acids and the acid number <2 mgKOH.g -1 ) and fatty acid profile (high content of monounsaturated fatty acids ≈72 w%) fit the requirements set in EN14214 biodiesel standard, regardless of the harvest date, harvest season can be extended to at least three months without compromising the oil quality for biodiesel production. Thus, intrinsic disservice of K. paniculata - high seed yielding can be shifted into a novel ecosystem service - quality biodiesel production. Aiming to avoid viable seed deposition in the ground and formation of the invasive seed bank, early August harvest can be recommended., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Metallothioneins are peculiar cysteine rich, heat resistant, small cellular plasma proteins expressed through almost all life forms. The currently established biological functions of metallothioneins are the homeostasis of essential metals and protection against toxic transitional metals (TM) alongside defence from oxidative stress by direct scavenging of reactive oxygen and nitrogen species (ROS and RNS). In mammals, among the four main evolutionary conserved forms, only the ubiquitously expressed metallothionein 1 and 2 (here abbreviated as MT) are inducible by TM, oxidative stress, glucocorticoids and starvation among various other stimuli. However, more than sixty years after being discovered, metallothioneins still bear unresolved issues about their possible physiological function and regulation. The biological function of MTs has still not been associated with the in vitro-demonstrated capacity of MT interaction with cellular molecules glutathione (GSH) or adenosine triphosphate (ATP), or with the possibility of direct iron-MT binding in the reducing intracellular environment of some organelles, e.g. lysosomes. Iron as the most abundant cellular TM is also one of the main physiological sources of ROS. Moreover, iron exhibits strain, sex and age differences that reflected ROS generation and MT induction in (patho)physiology and toxicology studies. A recent study showed that iron sex differences follows expression of both ferritin and MT leading to wide implications from essential TM interconnectivity to aging. This review places emphasis on biochemically proven but physiologically ignored interactions of MT with iron to stimulate advanced research for establishing a wide frame of the biological roles of MTs important for health and longevity.

This study was designed to evaluate the nano-bio interactions between endogenous biothiols (cysteine and glutathione) with biomedically relevant, metallic nanoparticles (silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs)), in order to assess the biocompatibility and fate of nanoparticles in biological systems. A systematic and comprehensive analysis revealed that the preparation of AgNPs and AuNPs in the presence of biothiols leads to nanoparticles stabilized with oxidized forms of biothiols. Their safety was tested by evaluation of cell viability, reactive oxygen species (ROS) production, apoptosis induction and DNA damage in murine fibroblast cells (L929), while ecotoxicity was tested using the aquatic model organism Daphnia magna . The toxicity of these nanoparticles was considerably lower compared to their ionic metal forms (i.e., Ag + and Au 3+ ). The comparison with data published on polymer-coated nanoparticles evidenced that surface modification with biothiols made them safer for the biological environment. In vitro evaluation on human cells demonstrated that the toxicity of AgNPs and AuNPs prepared in the presence of cysteine was similar to the polymer-based nanoparticles with the same core material, while the use of glutathione for nanoparticle stabilization was considerably less toxic. These results represent a significant contribution to understanding the role of biothiols on the fate and behavior of metal-based nanomaterials., (Copyright © 2019, Pem et al.; licensee Beilstein-Institut.)

Metallothioneins (MTs) exhibit binding affinity for several essential and toxic trace elements. Previous studies in rodents indicated sex differences in the hepatic and renal expression of MTs and concentrations of various elements. The mechanism responsible for these differences has not been resolved. Here, in the liver and kidney tissues of sham-operated and gonadectomized male and female rats we determined the expression of MT1 and MT2 (MT1&2) mRNA by RT-PCR, abundance of MT1&2 proteins by Western blotting and immunocytochemistry, concentrations of essential (Fe, Zn, Cu, Co) and toxic (Cd, Hg, Pb) elements by ICP-MS, and oxidative status parameters (SOD, GPx, MDA, GSH) by biochemical methods. In both organs, the expression of MT1&2 mRNA and MT1&2 proteins was female-dominant, upregulated by castration, and downregulated by ovariectomy. Concentrations of Fe in the liver and Co in the kidneys followed the same pattern. Most other elements (Zn, Cu, Cd, Hg) exhibited female- or male-dominant sex differences, affected by gonadectomy in one or both organs. Pb was sex- and gonadectomy-unaffected. GPx and MDA were elevated and associated with the highest concentrations of Fe only in the female liver. We conclude that the sex-dependent expression of MT1&2 mRNA and proteins in the rat liver and kidneys may include different mechanisms. In the liver, the female-dominant tissue concentrations of Fe may generate oxidative stress which is a potent enhancer of MTs production, whereas in kidneys, the female-dominant expression of MTs may be unrelated to Fe-mediated oxidative stress., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2

Rodent kidneys exhibit three isoforms of metallothioneins (MTs), MT1, MT2 and MT3, with poorly characterized localization along the nephron. Here we studied in adult male Wistar rats the renal expression of MTs mRNA by end-point RT-PCR and MT proteins by immunochemical methods The expression pattern of MT1 mRNA was cortex (CO)>outer stripe (OS)=inner stripe (IS)=inner medulla (IM), of MT2 mRNA was IM>CO>IS=OS, and of MT3 mRNA was IM>CO=OS=IM. MT1/2-antibody stained with heterogeneous intensity the cell cytoplasm and nuclei in proximal tubule (PT) and thin ascending limb, whereas MT3-antibody stained weakly the cell cytoplasm in various cortical tubules and strongly the nuclei in all nephron segments. However, the isolated nuclei exhibited an absence of MT1/2 and presence of MT3 protein. In MT1/2-positive PT cells, the intracellular staining appeared diffuse or bipolar, but the isolated brush-border, basolateral and endosomal membranes were devoid of MT1/2 proteins. In the lumen of some PT profiles, the heterogeneously sized MT1/2-rich vesicles were observed, with the limiting membrane positive for NHE3, but negative for V-ATPase, CAIV, and megalin, whereas their interior was positive for CAII and negative for cytoskeleton. They seem to be pinched off from the luminal membrane of MT1/2-rich cells, as also indicated by transmission electron microscopy. We conclude that in male rats, MTs are heterogeneously abundant in the cell cytoplasm and/or nuclei along the nephron. The MT1/2-rich vesicles in the tubule lumen may represent a source of urine MT and membranous material, whereas MT3 in nuclei may handle zink and locally-produced reactive oxygen species., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na(+)-K(+)-ATPase (EC 3.6.3.9), secondary active Na(+)-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na(+)-K(+)-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1-OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.

Cadmium (Cd)-intoxicated experimental animals exhibit impaired renal secretion of organic anions (OA) and cations (OC), indicating their transporters (Oats and Octs) in the proximal tubule (PT) basolateral membrane as possible targets of Cd. To correlate transport data from the literature with the expression of relevant transporters, we performed immunochemical and RT-PCR studies of renal Oats and Octs in the subchronic (treatment with CdCl2; 2 mg Cd/kg b.m./day, for 2 weeks) and acute (treatment with Cd-metallothionein (CdMT); 0.4 mg Cd/kg b.m., 6 or 12 h before killing) models of Cd nephrotoxicity. In the subchronic model, PT exhibited a minor loss of basolateral invaginations and overall unchanged expression of Na(+)/K(+)-ATPase and GAPDH proteins and mRNAs, while the expression of Oat and Oct proteins and their mRNAs was strongly downregulated. In the acute model, a time-related redistribution of basolateral transporters to the intracellular vesicular compartment was a major finding. However, 6 h following CdMT treatment, the total abundance of Oat and Oct proteins in the renal tissue remained unchanged, the expression of mRNAs decreased only for Oats, while a limited Oat1 and Na(+)/K(+)-ATPase immunoreactivity in the PT apical membrane indicated loss of cell polarity. As tested in rats treated with colchicine, the observed loss/redistribution of basolateral transporters in both models may be independent on microtubules. Therefore, the diminished renal secretion of OA and OC via PT in Cd nephrotoxicity may result from (a) limited loss of secretory surface (basolateral invaginations), (b) selective loss of Oats and Octs, and, ((c) loss of cell polarity.)

The proliferation of silver nanoparticle (AgNP) production and use owing to their antimicrobial properties justifies the need to examine the resulting environmental impacts. The discharge of biocidal nanoparticles to water bodies may pose a threat to aquatic species. This study evaluated the effects of citrate-coated AgNPs on the standardized test organism Daphnia magna Straus clone MBP996 by means of biochemical biomarker response. AgNP toxicity was compared against the toxic effect of Ag(+). The toxicity endpoints were calculated based upon measured Ag concentrations in exposure media. For AgNPs, the NOAEC and LOAEC values at 48 h were 5 and 7 μg Ag/L, respectively, while these values were 0.5 and 1 μg Ag/L, respectively, for Ag(+). The EC50 at 48 h was computed to be 12.4 ± 0.6 and 2.6 ± 0.1 μg Ag/L for AgNPs and Ag(+), respectively, with 95 % confidence intervals of 12.1-12.8 and 2.3-2.8 μg Ag/L, respectively. These results indicate significant less toxicity of AgNP compared to free Ag(+) ions. Five biomarkers were evaluated in Daphnia magna neonates after acute exposure to Ag(+) or AgNPs, including glutathione (GSH) level, reactive oxygen species (ROS) content, and catalase (CAT), acetylcholinesterase (AChE), and superoxide dismutase (SOD) activity. AgNPs induced toxicity and oxidative stress responses in D. magna neonates at tenfold higher concentrations than Ag. Biochemical methods revealed a clear increase in AChE activity, decreased ROS level, increased GSH level and CAT activity, but no significant changes in SOD activity. As Ag(+) may dissolve from AgNPs, these two types of Ag could act synergistically and produce a greater toxic response. The observed remarkably high toxicity of AgNPs (in the parts-per-billion range) to crustaceans indicates that these organisms are a vulnerable link in the aquatic food chain with regard to contamination by nanosilver. Graphical Abstract ᅟ.

Aim: To investigate whether the sex-dependent expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) changes in a rat model of ethylene glycol (EG)-induced hyperoxaluria., Methods: Rats were given tap water (12 males and 12 females; controls) or EG (12 males and 12 females; 0.75% v/v in tap water) for one month. Oxaluric state was confirmed by biochemical parameters in blood plasma, urine, and tissues. Expression of sat-1 and rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase 1 (Adh1) and hydroxy-acid oxidase 1 (Hao1), was determined by immunocytochemistry (protein) and/or real time reverse transcription polymerase chain reaction (mRNA)., Results: EG-treated males had significantly higher (in μmol/L; mean±standard deviation) plasma (59.7±27.2 vs 12.9±4.1, P<0.001) and urine (3716±1726 vs 241±204, P<0.001) oxalate levels, and more abundant oxalate crystaluria than controls, while the liver and kidney sat-1 protein and mRNA expression did not differ significantly between these groups. EG-treated females, in comparison with controls had significantly higher (in μmol/L) serum oxalate levels (18.8±2.9 vs 11.6±4.9, P<0.001), unchanged urine oxalate levels, low oxalate crystaluria, and significantly higher expression (in relative fluorescence units) of the liver (1.59±0.61 vs 0.56±0.39, P=0.006) and kidney (1.77±0.42 vs 0.69±0.27, P<0.001) sat-1 protein, but not mRNA. The mRNA expression of Adh1 was female-dominant and that of Hao1 male-dominant, but both were unaffected by EG treatment., Conclusions: An increased expression of hepatic and renal oxalate transporting protein sat-1 in EG-treated female rats could protect from hyperoxaluria and oxalate urolithiasis.

Detoxification of mercury (Hg) with selenium (Se) in the early postnatal period with regard to the expression of metallothionein protein (MT), essential element status, and lipid peroxidation level in tissues has not been studied. Seven-day-old Wistar pups were orally pretreated with Se [6 μmol Na2SeO3/kg body weight (b.w.)] for 3 days and then cotreated with Hg (6 μmol HgCl2/kg b.w.) for the following 4 days. This group (Se + Hg) was compared to the groups treated with Hg, Se, or vehicle (control). Compared to the Hg-group, Se + Hg-group exhibited lower renal MT expression, reduced accumulation of Hg, Cu and Zn, and reduced excretion of Se, Hg and Zn in urine. In the liver, MT was stimulated by Se treatment in both, Se and Se + Hg-group. Hepatic and brain levels of the endogenous essential elements Cu, Fe, Mg, and Zn remained unchanged in all of the studied groups. Brain Hg levels and oxidation of lipids measured as thiobarbituric acid reactive substances were diminished in Se + Hg-group of pups compared to the Hg-group. This study suggests that Se pretreatment can help reduce Hg in the tissues of suckling rats, simultaneously preventing impairment of essential element levels in the kidneys and their excessive excretion via urine. Also, Se was shown to prevent oxidative damage of lipids in the brain, which is particularly susceptible to Hg during the early postnatal period.

In the mammalian kidney, nonglycosylated and glycosylated forms of aquaporin protein 1 (AQP1) coexist in the luminal and basolateral plasma membranes of proximal tubule and descending thin limb. Factors that influence AQP1 expression in (patho)physiological conditions are poorly known. Thus far, only angiotensin II and hypertonicity were found to upregulate AQP1 expression in rat proximal tubule in vivo and in vitro (Bouley R, Palomino Z, Tang SS, Nunes P, Kobori H, Lu HA, Shum WW, Sabolic I, Brown D, Ingelfinger JR, Jung FF. Am J Physiol Renal Physiol 297: F1575-F1586, 2009), a phenomenon that may be relevant for higher blood pressure observed in men and male experimental animals. Here we investigated the sex-dependent AQP1 protein and mRNA expression in the rat kidney by immunochemical methods and qRT-PCR in tissue samples from prepubertal and intact gonadectomized animals and sex hormone-treated gonadectomized adult male and female animals. In adult rats, the overall renal AQP1 protein and mRNA expression was ∼80% and ∼40% higher, respectively, in males than in females, downregulated by gonadectomy in both sexes and upregulated strongly by testosterone and moderately by progesterone treatment; estradiol treatment had no effect. In prepubertal rats, the AQP1 protein expression was low compared with adults and slightly higher in females, whereas the AQP1 mRNA expression was low and similar in both sexes. The observed differences in AQP1 protein expression in various experiments mainly reflect changes in the glycosylated form. The male-dominant expression of renal AQP1 in rats, which develops after puberty largely in the glycosylated form of the protein, may contribute to enhanced fluid reabsorption following the androgen- or progesterone-stimulated activities of sodium-reabsorptive mechanisms in proximal tubules., (Copyright © 2015 the American Physiological Society.)

In this study, the polyphenolic profile of 11 Morus alba fruits grown in the Vojvodina region was investigated. Ultra high performance liquid chromatography (UHPLC) coupled with Linear Trap Quadrupole and OrbiTrap mass analyzer, and UHPLC coupled with a diode array detector and a triple-quadrupole mass spectrometer were used for the identification and quantification of the polyphenols, respectively. A total of 14 hydroxycinnamic acid esters, 13 flavonol glycosides, and 14 anthocyanins were identified in the extracts with different distributions and contents according to the sampling. The total phenolic content ranged from 43.84 to 326.29 mg GAE/100g frozen fruit. The radical scavenging capacity (50.18-86.79%), metal chelating ability (0.21-8.15%), ferric ion reducing power (0.03-38.45 μM ascorbic acid) and superoxide anion radical scavenging activity (16.53-62.83%) were assessed. The findings indicated that mulberry polyphenolics may act as potent superoxide anion radical scavengers and reducing agents., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mycotoxin ochratoxin A (OTA) is nephrotoxic in various animal species. In rodents, OTA intoxication impairs various proximal tubule (PT) functions, including secretion of p-aminohippurate (PAH), possibly via affecting the renal organic anion (OA) transporters (Oat). However, an effect of OTA on the activity/expression of specific Oats in the mammalian kidney has not been reported. In this work, male rats were gavaged various doses of OTA every 2nd day for 10 days, and in their kidneys we studied: tubule integrity by microscopy, abundance of basolateral (rOat1, rOat3) and brush-border (rOat2, rOat5) rOat proteins by immunochemical methods, and expression of rOats mRNA by RT-PCR. The OTA treatment caused: a) dose-dependent damage of the cells in S3 segments of medullary rays, b) dual effect upon rOats in PT: low doses (50-250 microg OTA/kg b.m.) upregulated the abundance of all rOats, while a high dose (500 microg OTA/kg b.m.) downregulated the abundance of rOat1, and c) unchanged mRNA expression for all rOats at low OTA doses, and its downregulation at high OTA dose. Changes in the expression of renal Oats were associated with enhanced OTA accumulation in tissue and excretion in urine, whereas the indicators of oxidative stress either remained unchanged (malondialdehyde, glutathione, 8-hydroxydeoxyguanosine) or became deranged (microtubules). While OTA accumulation and downregulation of rOats in the kidney are consistent with the previously reported impaired renal PAH secretion in rodents intoxicated with high OTA doses, the post-transcriptional upregulation of Oats at low OTA doses may contribute to OTA accumulation and development of nephrotoxicity.

To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and "masks" the antibody binding sites (epitopes). In order to "unmask" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed in vivo or in vitro. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed in vivo, tissue fixed in vitro exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach.

The renal reabsorption and/or excretion of various organic anions is mediated by specific organic anion transporters (OATs). OAT2 (Slc22a7) has been identified in rat kidney, where its mRNA expression exhibits gender differences [females (F) > males (M)]. The exact localization of OAT2 protein in the mammalian kidney has not been reported. Here we studied the expression of OAT2 mRNA by RT-PCR and its protein by Western blotting (WB) and immunocytochemistry (IC) in kidneys of adult intact and gonadectomized M and F, sex hormone-treated castrated M, and prepubertal M and F rats, and the protein in adult M and F mice. In adult rats, the expression of OAT2 mRNA was predominant in the outer stripe (OS) tissue, exhibiting 1) gender dependency (F > M), 2) upregulation by castration and downregulation by ovariectomy, and 3) strong downregulation by testosterone and weak upregulation by estradiol and progesterone treatment. A polyclonal antibody against rat OAT2 on WB of isolated renal membranes labeled a approximately 66-kDa protein band that was stronger in F. By IC, the antibody exclusively stained brush border (BB) of the proximal tubule S3 segment (S3) in the OS and medullary rays (F > M). In variously treated rats, the pattern of 66-kDa band density in the OS membranes and the staining intensity of BB in S3 matched the mRNA expression. The expression of OAT2 protein in prepubertal rats was low and gender independent. In mice, the expression pattern largely resembled that in rats. Therefore, OAT2 in rat (and mouse) kidney is localized to the BB of S3, exhibiting gender differences (F > M) that appear in puberty and are caused by strong androgen inhibition and weak estrogen and progesterone stimulation.

SGLT1 (SLC5A1) mediates a part of glucose and galactose reabsorption in the mammalian proximal tubule (PT), but the detailed localization of the transporter along the tubule is still disputable. Here, we used several methods to localize rat SGLT1 (rSGLT1) in the kidneys of intact and variously treated male (M) and female (F) rats. In immunoblots of isolated cortical (C) and outer stripe (OS) brush-border membranes (BBM), a peptide-specific polyclonal antibody for rSGLT1 labeled a sharp inzone-, and gender-dependent approximately 40-kDa protein and a broad approximately 75-kDa band that exhibited strong zonal (OS > C) and gender differences (F > M). In tissue cryosections, the antibody strongly stained BBM of the S3 PT segments in the OS and medullary rays (F > M) and smooth muscles of the blood vessels and renal capsule (F approximately M) and weakly stained the apical domain of other PT segments in the C (F approximately M). The phlorizin-sensitive uptake of d-[(3)H]galactose in BBM vesicles, as well as the tissue abundance of rSGLT1-specific mRNA, matched the immunoblotting data related to the 75-kDa protein and the immunostaining in S3, proving zonal and gender differences in the functional transporter. Ovariectomy had no effect, castration upregulated, whereas treatment of castrated rats with testosterone, but not with estradiol or progesterone, downregulated the 75-kDa protein and the immunostaining in S3. We conclude that in the rat kidney, the expression of SGLT1 is represented by a 75-kDa protein localized largely in the PT S3 segments, where it exhibits gender differences (F > M) at both the protein and mRNA levels that are caused by androgen inhibition.

The use of renal cortical slices in vitro and the data obtained in these studies have been subjects of controversy, largely due to uncertain viability, e.g., structural and functional integrity of the proximal and other tubules. However, detailed studies of tubule integrity have not been reported. To correlate functional and structural viability of the hand-cut rat renal cortical slices, incubated in optimally conditioned media for up to 25 h, we studied the time course of p-aminohippurate (PAH) uptake, the immunocytochemical distribution of several proteins that reside in the proximal tubule basolateral [Na/K-ATPase, organic anion transporters (OAT)1 and OAT3], or brush border [megalin, sodium-proton exchanger (NHE)3] membrane, as well as the general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules). PAH uptake in slices was proportional to time within 1 h of incubation and gradually declined thereafter. The immunostaining experiments indicated a fast, time-dependent loss of basolateral transporters, at a rate of OAT1 > Na/K-ATPase > OAT3. In the brush border membrane, the loss of megalin was faster than that of NHE3, and a partial redistribution of NHE3 into the basolateral domain indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in the proximal tubule was already visible after 15 min of incubation and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated a compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in the proximal tubule epithelium, with sloughing of brush border and cell debris into the tubule lumen, detachment of cells from the basal membrane, and opening and widening of the tubule lumen. We conclude that the loss of cellular structure, cytoskeleton, and cell membrane transporters in the nephron epithelium is a very early event in the incubated rat renal cortical slices.