Determination Of Enzyme Activity Research Articles

THE GLA D313Y MUTATION IN FABRY DISEASE: DIAGNOSTIC DILEMMAS AND THERAPEUTIC CONSIDERATIONS

Abstract Fabry disease is a genetic disorder caused by mutations in the GLA gene, which encodes alpha–galactosidase A, leading to enzyme dysfunction and subsequent accumulation of glycosphingolipids. In the classical form, it occurs in childhood or early adolescence with renal, cardiac, and neurological involvement, resulting in a poor prognosis. The GLA D313Y is a missense mutation resulting in a significant reduction of enzyme activity in plasma, while leukocyte enzyme activity remains within normal limits, leading to a "pseudo–deficiency" of enzyme activity with uncertain pathogenicity. In literature, it has been observed that individuals with this mutation may present predominantly with central neurologic manifestations, characterized by multifocal white matter lesions. Historically they do not receive indications for initiating enzyme replacement or molecular chaperone therapies. We present the case of a man affected by arterial hypertension and history of chronic lower limb pain who experienced a lacunar stroke at the age of 35. The GLA D313Y mutation was identified and enzyme activity assay in plasma was within normal limits, precluding the initiation of enzyme replacement therapy. Over the following years, he suffered two additional ischemic strokes, a progressive decline in renal function to stage 3, and acute coronary syndrome. Transthoracic echocardiography revealed hypertrophic non–obstructive cardiomyopathy (SIVd 20 mm) with normal biventricular ejection fraction. Cardiac magnetic resonance showed late gadolinium enhancement of inferior junctional and inferior wall in middle segment (Fig.1). Considering the patient‘s clinical conditions involving multi–organ impairment, an enzymatic activity test was performed on leukocytes, revealing a reduced activity. This prompted the decision to initiate enzyme replacement therapy. This case report supports the hypothesis that the D313Y mutation may be associated with a later presentation and not only neurological involvement. In our experience, it’s mandatory to proceed with the determination of leukocyte enzyme activity in presence of normal enzyme activity in plasma. As an alternative, the detection of glycosphingolipids’ accumulation could be useful, but myocardial and renal biopsy poses a high bleeding risk, especially in anticoagulated or antiaggregated patients. The authors propose considering the initiation of therapy in patients with the D313Y mutation who exhibit significant organ involvement.

Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and ∼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 μM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 μM) and mGPDH (Ki 54.70 μM) activities. siRNAs transfection showed ∼50 % and ∼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Effect of the Oligosaccharid Fraction of Fusarium oxysporum f. sp. vasinfectum on the Activity of Phenolic Metabolism Enzymes in Cotton Leaves

The synthesis of secondary metabolites, notably phenolic compounds, is part of the plant's defense reactions. The enzymes of phenolic metabolism can constitute a parameter of measurement of the defense of the plants. The aim of this work is to evaluate the activity of phenolic metabolism enzymes and an antioxidant enzyme after treatment of cotton plants with a fungal elicitor. Cotton plants obtained from the cultivar Y764AG3 were treated with the oligosaccharide fraction (FOS) of Fusarium oxysporum f. sp. Vasinfectum (FOV). The extraction of the enzymes is carried out cold by grinding the leaves in extraction buffer in the presence of PVP (0.5 %) and 0.1 M sodium phosphate buffer. The supernatant obtained after centrifugation of the filtrate was purified with Dowex-2 in order to obtain the purified enzymatic fraction. This fraction was used for the determination of enzyme activity by reading the absorbances. The results showed that the activity of ammonia-lyase activity was higher in the treated plants than in the control. This reflects an increase in the biosynthesis of phenolic compounds in the treated plants. On the other hand, oxidoreductase activity was higher in control plants than in treated plants. This indicates a decrease in the degradation of phenolic compounds in the treated plants. This study revealed the existence of an active phenolic metabolism in the FOS-treated plants. Thus defence mechanisms are activated in plants treated with the oligosaccharide fraction of Fusarium oxysporum f. sp. Vasinfectum.

N-succinyl-chitosan as ecofriendly pesticide carriers: Nano encapsulation and synergistic antifungal effect on 4-hydroxyphenyl-2-propenyl-1-one derivatives based on chalcone structure

IntroductionTraditional pesticides have poor-water solubility, high toxicity and low bioavailability. Therefore, it is of great significance for the sustainable and healthy development of the pesticide industry to develop efficient and ecofriendly new chemical pesticide products and formulations. ObjectivesThis study aims to synthesize a series of derivatives based on chalcone structure (HPPO), and then use the amphiphilic and self-assembly characteristics of N-succinyl-chitosan (NSCS) to prepare HPPO@NSCS nanoparticles (HPPO@NSCS NPs) in order to realize the green application of HPPO, and investigate the antifungal activity and mechanisms of HPPO@NSCS NPs. MethodsNSCS was synthesized by structural modification using chitosan as the carrier. Based on its amphiphilic and self-assembly characteristics, HPPO-16@NSCS NPs were reasonably prepared by combining with active small molecule HPPO-16. Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), fluorescence spectroscopy (FS) and high-performance liquid chromatography (HPLC) were used to characterize the physicochemical properties of NSCS and HPPO-16@NSCS NPs. The inhibitory activity of nanopesticides against Rhizoctonia solani (R. solani) was tested in vivo and in vitro. The mechanism of antifungal action was discussed from the observation of pathogen morphology, fluorescence staining and enzyme activity determination. Results28 small molecules based on chalcone structure (HPPO-1-28), NSCS and HPPO-16@NSCS were successfully synthesized. The application of HPPO-16@NSCS could impair the development, cell structure, cellular energy utilization, and metabolism pathways of the fungi. The protective effects of HPPO-16@NSCS NPs on rice leaves and leaf sheaths were 80.9 and 76.1 %, respectively, which were better than those of azoxystrobin. ConclusionThis study reveals that these simple chalcone derivatives can be further explored as viable antibacterial alternatives and NSCS as a novel pesticide matrix can be used for the delivery of more insoluble pesticides.

Changes in growth, morphology, and levels of digestive enzymes and growth-related hormones in early ontogeny of black scraper, Thamnaconus modestus

IntroductionThe black scraper, Thamnaconus modestus, is a highly valued marine fish species, but its output has gradually decreased in recent years, which may be due to its low survival rate during early ontogenesis.MethodsTherefore, in this study, we assessed the changes in growth, morphology, digestive enzymes, and hormone levels in T. modestus from 0–60 days post-hatching (dph) and revealed growth turning points by morphological measurement and determination of digestive enzyme activities and hormone levels. We found that ontogenesis could be divided into the larval (0–20 dph) and juvenile (20–60 dph) stages. Acid and alkaline protease activity significantly increased and decreased, respectively, from 12–25 dph, likely due to the development of stomach and gastric glands. Acid phosphatase levels significantly increased at 0 and 4 dph, which may be related to the regulation of metabolism and immune protection. A sharp increase in alkaline phosphatase levels at 20 and 25 dph was observed and was likely due to the development of the brush border membrane of enterocytes. The amylase level was significantly higher at 25, 30, and 35 dph, possibly due to better digestion and absorption during the transition from consuming Artemia to compound feed. In newly hatched larvae, the level of thyroid hormones triiodothyronine (T3) and thyroxine (T4) gradually increased and peaked at 35 dph, highlighting the importance of these hormones during the development of T. modestus. Growth hormone (GH) levels first increased from 0–8 dph, with a plateau at 8–20 dph, and then increased at 25–30–35 dph. For insulin-like growth factor 1 (IGF-1), a significant increase with a subsequent plateau was observed between 8 and 20 dph, followed by a substantial decrease between 30 and 35 dph. These results suggest that the regulating functions of GH and IGF-1 are synchronised. Digestive enzyme activity and hormone levels of abnormal fry at 30 dph were lower than those of normal fish, highlighting the importance of specific hormones, especially T4 and IGF-1, in the development of T. modestus.

Glucose Catabolite Repression Participates in the Regulation of Sialidase Biosynthesis by Antarctic Strain Penicillium griseofulvum P29.

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.

WITHDRAWN: Isolation and Functional Study of Efficient Cellulose Degrading Bacteria from Horse Cecum Contents

ObjectivesAnimals that consume and digest roughage for a long time have unique gut microbiota in their gastrointestinal tract. Studying the isolation of beneficial functional bacteria from the gut can provide resources for the development of feed microbial additives. Therefore, the purpose of this study was to isolate and identify bacteria with cellulose degradation ability from the cecal contents of Yili horses, and to provide resources for the development of feed microbial additives. MethodsFirstly, the cecal contents of 18 Yili horses were collected, and cellulose degrading bacteria were isolated and screened using CMC-Na screening medium under aerobic and anaerobic conditions. The strains with strong cellulose degradation ability were identified using Congo red staining and DNS methods. The top 60 strains with enzyme index were selected, and full-length 16S rRNA sequencing technology was used to identify and construct a phylogenetic tree. Select the top 5 strains of bacteria with high enzyme activity based on the measured cellulase activity, and the sheep were treated with mixed bacterial suspension by gavage. The rumen microbiota of sheep was analyzed using 16S rDNA amplicon sequencing technology. ResultsA total of 263 single colonies were isolated, of which 120 single colonies had a hydrolysis circle and colony enzymatic index (EI) of >1.5. Enzyme activity determination screening of the top 60 strains of full-length 16S rRNA sequencing, sequencing results show that the strains are mostly Bacillus spp. Among the 60 strains of bacteria, CE207 has the highest enzyme activity of 14.81 U/mL, and the lowest enzyme activity of CE105 is 1.91 U/mL. The mixed bacteria significantly increased the daily weight gain of sheep (P < 0.001) and feed intake (P < 0.001), and feed conversion rate increased from 7.75% to 10.09%. Sequencing results of sheep rumen microorganisms showed that, compared with the control group, the experimental group contained Muribaculum, Parvibacter, Desulfovibrio, Novosphingobium, Cutibacterium, Parasutterella, Lachnospiraceae_UCG-006, and Allobaculum genera increased significantly in abundance, and Fibrobacter and NK4A214_group genera decreased significantly in abundance, suggesting that a mixture of bacteria can influence the changes in the microflora of the rumen intestinal tract of sheep. ConclusionsThis finding suggests that the cellulose degrading bacteria isolated in the study can effectively promote the growth and development of herbivores such as sheep, and that efficient cellulose degrading bacteria in the animal gut may become a new direction for the development of feed additives in the future.

Integrated metabolic phenotypes and gene expression profiles reveal enzymatic browning mechanism in postharvest chrysanthemum

Chrysanthemum (Chrysanthemum morifolium Ramat) is a valuable crop mainly used for medicine, beverages and ornament. Enzymatic browning causes serious postharvest flower deterioration of chrysanthemum, but the mechanism remains unclear. To clarify the mechanism involved, the cellular ultrastructure, metabolites, browning products, and differentially expressed genes of chrysanthemum were studied systematically. During the browning process, cellular compartmentalization was severely disrupted and the contents of chlorogenic acid derivatives and flavonoid glycosides decreased significantly. A browning product with a dihydrobenzofuran skeleton was purified and identified for the first time from a simulated browning reaction of chlorogenic acid catalyzed by peroxidase. Moreover, six differentially expressed genes were for the first time identified to be involved in the regulation of oxidase by transcriptome and qPCR, and one of which (c76073_g1) was validated using in vitro enzyme activity determination. This research elucidates the inherent mechanism of enzymatic browning, provides a scientific explanation for the steam-treated processing of chrysanthemums and lays the foundation for the molecular breeding of anti-browning chrysanthemum varieties.

Non-canonical amino acids uncover the significant impact of Tyr671 on Taq DNA polymerase catalytic activity.

Responsible for synthesizing the complementary strand of the DNA template, DNA polymerase is a crucial enzyme in DNA replication, recombination and repair. A highly conserved tyrosine (Tyr), located at the C-terminus of the O-helix in family A DNA polymerases, plays a critical role in enzyme activity and fidelity. Here, we combined the technology of genetic code extension to incorporate non-canonical amino acids and molecular dynamics (MD) simulations to uncover the mechanisms by which Tyr671 impacts substrate binding and conformation transitions in a DNA polymerase from Thermus aquaticus. Five non-canonical amino acids, namely l-3,4-dihydroxyphenylalanine (l-DOPA), p-aminophenylalanine (pAF), p-acetylphenylalanine (pAcF), p-cyanophenylalanine (pCNF) and p-nitrophenylalanine (pNTF), were individually incorporated at position 671. Strikingly, Y671pAF and Y671DOPA were active, but with lower activity compared to Y671F and wild-type. Y671pAF showed a higher fidelity than the Y671F, despite both possessing lower fidelity than the wild-type. Metadynamics and long-timescale MD simulations were carried out to probethe role of mutations in affecting protein structure, including open conformation, open-to-closed conformation transition, closed conformation, and closed-to-open conformation transition. The MD simulations clearly revealed that the size of the 671 amino acid residue and interactions with substrate or nearby residues were critical for Tyr671 to determine enzyme activity and fidelity.

Quantitative mass spectrometry with ¹⁸O labelling as an alternative approach for determining protease activity: an example of trypsin

SCIENTIFIC RELEVANCE. In the quality control of proteolytic enzyme components of medicinal products, the activity of proteases is determined by spectrophotometry, which involves mea­suring the amidase or esterase activity using a synthetic substrate and the proteolytic activity using the Anson method. These methods require special substrates and have low sensitivity; their specificity may be insufficient, which may lead to serious errors. Quantitative mass spectrometry is an alternative approach to protease activity assays, which involves adding an isotope-labelled peptide to hydrolysates of the test enzyme. This approach allows determining the activity of proteases, notably, by the hydrolysis of specific peptide bonds, while simulta­neously confirming the identity and specificity of the test sample. Quantitative mass spectrometry has high sensitivity and does not require special substrates.AIM. This study aimed to investigate the possibility of enzymatic activity assay and enzyme identification by quantitative mass spectrometry with ¹⁸O labelling through an example of trypsin with casein.MATERIALS AND METHODS. The study used trypsin, casein, and H₂¹⁸O (Izotop, Russia). Peptide separation was performed using an Agilent 1100 HPLC system; mass spectra were obtained using a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer. Quantitative mass spectrometry was performed using a standard peptide, which was obtained from casein by tryptic digestion and HPLC purification. For ¹⁸O labelling, the authors dried the peptide and incubated it in H₂¹⁸О water. The quantitative analysis of the product was carried out using MALDI-TOF mass spectrometry. The authors used quantitative mass spectrometry with ¹⁸O labelling to determine enzymatic activity and calculate the Michaelis constant (KM).RESULTS. Following the tryptic digestion of casein, the authors identified the fragments corre­sponding to casein chains. The authors produced the isotope-labelled standard peptide and calculated its concentration using mass spectrometry. The authors determined the rate of casein digestion by trypsin and calculated the KM for trypsin, which was 13.65±0.60 μM. The standard deviation for repeated measurements showed that the mass-spectrometric method had a lower error of measurement than the spectrophotometric method. The sensitivity threshold for the mass-spectrometric method was 0.50±0.08 μM.CONCLUSIONS. The results obtained with trypsin confirm the possibility of enzymatic activity determination by the proposed method of quantitative mass spectrometry with ¹⁸O labelling. According to the sensitivity evaluation results, this method can be used for the simultaneous determination of enzyme activity, identity, and specificity. The proposed mass spectrometry approach is universal, it does not require expensive materials and reagents, and it can be easily adapted to determine the activity of virtually any protease.

Targeted Screening of Fiber Degrading Bacteria with Probiotic Function in Herbivore Feces.

Cellulolytic bacteria with probiotic functions play a crucial role in promoting the intestinal health in herbivores. In this study, we aimed to correlate the 16S rRNA gene amplicon sequencing and fiber-degrading enzyme activity data from six different herbivore feces samples. By utilizing the separation and screening steps of probiotics, we targeted and screened high-efficiency fiber-degrading bacteria with probiotic functions. The animals included Maiwa Yak (MY), Holstein cow (CC), Tibetan sheep (TS), Southern Sichuan black goat (SG), Sichuan white rex rabbit (CR), and New Zealand white rabbit (ZR). The results showed that the enzymes associated with fiber degradation were higher in goat and sheep feces compared to cattle and rabbit's feces. Correlation analysis revealed that Bacillus and Fibrobacter were positively correlated with five types of fiber-degrading related enzymes. Notably, the relative abundance of Bacillus in the feces of Tibetan sheep was significantly higher than that of other five herbivores. A strain TS5 with good cellulose decomposition ability from the feces of Tibetan sheep by Congored staining, filter paper decomposition test, and enzyme activity determination was isolated. The strain was identified as Bacillus velezensis by biological characteristics, biochemical analysis, and 16S rRNA gene sequencing. To test the probiotic properties of Bacillus velezensis TS5, we evaluated its tolerance to acid and bile salt, production of digestive enzymes, antioxidants, antibacterial activity, and adhesion ability. The results showed that the strain had good tolerance to pH 2.0 and 0.3% bile salts, as well as good potential to produce cellulase, protease, amylase, and lipase. This strain also had good antioxidant capacity and the ability to antagonistic Staphylococcus aureus BJ216, Salmonella SC06, Enterotoxigenic Escherichia coli CVCC196, and Escherichia coli ATCC25922. More importantly, the strain had good self-aggregation and Caco-2 cell adhesion rate. In addition, we tested the safety of Bacillus velezensis TS5 by hemolysis test, antimicrobial susceptibility test, and acute toxicity test in mice. The results showed that the strain had no hemolytic phenotype, did not develop resistance to 19 commonly used antibiotics, had no cytotoxicity to Caco-2, and did not have acute toxic harm to mice. In summary, this study targeted isolated and screened a strain of Bacillus velezensis TS5 with high fiber-degrading ability and probiotic potency. This strain can be used as a potential probiotic for feeding microbial preparations for ruminants.

Identification and Characterization of Neuroprotective Properties of Thaumatin-like Protein 1a from Annurca Apple Flesh Polyphenol Extract.

Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial neurodegenerative disorders that are mostly treated with drugs inhibiting key enzymes of cholinergic and aminergic neurotransmission, such as acetyl and butyryl cholinesterase (AChE, BuChE) or monoamine oxidases (MAO)-A/B, and of Aβ1-40 aggregation. Diet plant components with multitarget functions are promising compounds in the prevention of AD and PD. Our aim was to identify neuroprotective compounds from Annurca apple polyphenol extract (AFPE). AFPE was fractionated by gel filtration, and the eluted peaks were subjected to chemical analyses (i.e., RP-HPLC and mass spectrometry), determination of inhibitory enzyme activity and cell effects by MTT, and morphology assays. In AFPE, we identified thaumatin-like protein 1a, belonging to the pathogenesis-related protein (PR) family. This protein showed the best inhibitory activity on AChE, MAO-A (IC50 = 5.53 µM and 1.71 µM, respectively), and Aβ1-40 fibril aggregation (IC50 = 9.16 µM), compared to AFPE and other polyphenol-containing fractions. Among the latter, Peak 4 reverted Aβ fibril formation (IC50 = 104.87 µM). Moreover, thaumatin-like protein 1a protected AGS and MKN-28 cells from serum-deprivation-induced stress conditions. We showed that AFPE exerted neuroprotective functions not only through its polyphenols but also through thaumatin-like protein 1a, which acted like a multitarget molecule.

Bio-augmentation and bio-stimulation with kenaf core enhanced bacterial enzyme activities during bio-degradation of petroleum hydrocarbon in polluted soil

Indigenous micro-organisms often possess the ability to degrade petroleum hydrocarbon (PHC) in polluted soil. However, this process can be improved by supplementing with nutrients or the addition of more potent microbes. In this study, the ability of kenaf-core to stimulate the PHC degradation capability of microbial isolates from PHC polluted soil samples was evaluated. The standard experimental methods used in this study include: the digestion and analysis of the physico-chemical properties of petroleum hydrocarbon contaminated and non-contaminated soil samples; evaluation of petroleum hydrocarbon biodegradation using bio-augmentation and bio-stimulation (with kenaf-core) treatments; and, determination of soil microbial enzyme activities. Results from this study show that K, Na, total nitrogen, organic carbon, exchangeable cations, and heavy metals were found to be significantly (P < 0.05) higher in the polluted soil than in the non-polluted soil. Also, the polluted samples had pH values ranging from 5.5 to 6.0 while the non-polluted samples had a pH of 7.6. The microbial enzyme activities were comparatively lower in the polluted soils as compared to the non-polluted soil. The percentage degradation in the kenaf-core treated samples (AZ1T2—78.38; BN3T2—70.69; OL1T2—71.06; OT1T2—70.10) were significantly (P < 0.05) higher than those of the untreated (AZ1T1—13.50; BN3T1—12.50; OL1T1—10.55; OT1T1—9.50). The degradation of petroleum hydrocarbon in the bio-augmented and bio-stimulated treatments increased with increasing time of incubation, and were higher than that of the untreated sample. Comparatively, the treatment with a combination of kenaf-core and rhamnolipid exhibited a significantly (P < 0.05) higher degradation rate than that of the treatment with only kenaf core or rhamnolipid. While, the bio-stimulated and bio-augmented treatments had appreciable microbial counts that are higher than that of the untreated. In conclusion, the nutrient-supplement with kenaf-core significantly enhanced microbial growth and activities in the soil, thus improving their ability to biodegrade petroleum hydrocarbons in the polluted soils. Thus, supplementing with Kenaf core to encourage microbiological degradation of petroleum hydrocarbon is recommended.

Ameliorative effects of berberine and selenium against paracetamol-induced hepatic toxicity in rats.

Paracetamol (PCM) overdosing induces hepatotoxicity, which can result in death if the dose is high enough and the patients are not given N-acetyl cysteine. Berberine (BBR) has a variety of biological proprieties including anti-inflammatory and antioxidant activities. Assessment of the potential effect of BBR and selenium when used alone or together on the PCM-induced acute hepatic toxicity in rats. This research involved 40 clinically healthy mature adult male albino rats, their weights ranged from 150 to 200 g and housed in standard conditions. Our study involved evaluating the potential effect of BBR and selenium when used alone or together on the PCM-induced acute hepatic toxicity via estimation of the liver function tests, determination of the antioxidant enzyme activities, lipid peroxidation markers, immune-modulatory effects, liver histopathological, and immunohistochemical studies. Co-treatment of BBR (150 mg/kg BW) with selenium (5 mg/kg BW) showed significant improvement in the liver function parameters, the antioxidant enzyme activities, reduction in the nitric oxide (NO), lysozyme, malondialdehyde (MDA), TNF-α, and TGF-β1 levels, and marked elevation in the IgM levels. Altogether, BBR, selenium, or both augment antioxidant activity and alleviate PCM-induced hepatic toxicity.

Revisiting the urease production of MICP-relevant bacterium Sporosarcina pasteurii during cultivation

The ureolytic bacterium Sporosarcina pasteurii is commonly used for Microbial Induced Calcium Carbonate Precipitation (MICP). This process has a variety of applications in the fields of construction, geotechnical engineering, and environmental remediation. However, the factors influencing urease activity of S. pasteurii during cultivation are not yet fully understood. Even though there is debate over whether higher urease activity is in fact beneficial for MICP applications, knowledge about urease production can help provide ureolytic biomass more cost-effectively, reducing overall production costs. This study revisits the effect of various cultivation conditions on growth and urease activity by using an automated high-throughput microbioreactor system combined with a novel system for high-throughput urease activity quantification. This experimental set-up allows for unprecedented data density and enables new insights into urease production of S. pasteurii.Several factors were tested, including oxygen limitation, the feeding of urea, choice of ammonium salt, the proportion of ammonium salt in the medium, and lowering the pH during cultivation; none of these had a notable impact on urease production. Only the addition of a broad spectrum of nutrients through regular feeding with highly concentrated yeast extract and glucose led to a 70 % increased specific urease activity compared to a batch culture.These results can improve the understanding of the regulatory mechanisms governing urease expression in S. pasteurii during cultivation, and allow to adapt the cultivation process accordingly. Additionally, the new system for automated high-throughput enzyme activity determination presented in this study could be applied to optimize bioprocesses involving other ion producing enzymes.

Enzymatic easing of adhesion in honeydew-contaminated cotton for textile applications

Purpose The textile sector struggles with cotton stickiness from honeydew contamination. It hurts agriculture and marketability. This study aims to examine how bacterial enzymes could reduce honeydew-contaminated cotton adherence in textile businesses sustainably. Design/methodology/approach Enzyme was extracted from bacteria isolated from the fermented bamboo shoots “Lung siej”. The enzyme was tested for α-glucosidase using p-nitrophenyl-α-D-glucopyranoside as a substrate. Design of experiments determined enzyme activity temperature and reaction time. Laboratory-prepared artificial honeydew was added to ginning mill cotton to show honeydew contamination. After enzyme treatment, sticky cotton was tested for microscopic examination, ultraviolet (UV), Benedict’s, Elsner colorimetric, high volume instrument (HVI) and viscosity tests. Findings The bacterial isolate is characterized as Lysinibacillus sp. as confirmed by 16S rRNA gene sequencing. The enzyme extracted was identified as α-glucosidase. The ideal temperature and reaction time for enzymatic activity were 32 °C and 35 min, respectively, using central composite design. The microscopic examination, UV test, Benedict’s test, Elsner colorimetric test, HVI test and viscosity test showed that bacterial enzyme treatment reduced cotton fiber adherence. Originality/value Although few patents have examined the effect of yeast enzymes, to the best of the authors’ knowledge, a bacterial enzyme is investigated for the first time to reduce the adhesion of honeydew-contaminated cotton.

Stereoselective cardiotoxic effects of metconazole on zebrafish (Danio rerio) based on AGE-RAGE signalling pathway

Metconazole (MEZ) is a novel chiral triazole fungicide that is widely used to prevent and control soil-borne fungal pathogens and other fungal diseases. However, it has a long half-life in aquatic environments and thus poses potential environmental risks. This study evaluates the acute and stereoselective cardiotoxicity of MEZ in zebrafish (Danio rerio) embryos. In addition, transcriptomics, real-time quantitative PCR, enzyme activity determination, and molecular docking are performed to evaluate the molecular mechanisms underlying the cardiotoxicity of MEZ in zebrafish. MEZ decreases the heart rate while increasing the pericardial oedema rate; additionally, it induces stereoselective cardiotoxicity. 1S,5S-MEZ exhibits stronger cardiotoxicity than 1R,5R-MEZ. Furthermore, MEZ increases the expression of Ahr-associated genes and the transcription factors il6st, il1b, and AP-1. Heart development-related genes, including fbn2b, rbm24b, and tbx20 are differentially expressed. MEZ administration alters the activities of catalase, peroxidase, and glutathione-S-transferase in zebrafish larvae. Molecular docking indicates that 1R,5R-MEZ binds more strongly to the inhibitor-binding sites of p38 in the AGE-RAGE signalling pathway than to other MEZ enantiomers. Studies conducted in vivo and in silico have established the enantioselective cardiotoxicity of MEZ and its underlying mechanisms, highlighting the need to evaluate the environmental risk of chiral MEZ in aquatic organisms at the enantiomeric level.

Expression pattern and cellular localization of pepsinogen in early development and induced by different diets in the spotted knifejaw (Oplegnathus punctatus)

To solve the high mortality rate of early-stage larval feed conversion during aquaculture in Oplegnathus punctatus, the investigation of the structural and functional characteristics of the gastric tissue was conducted. Histological results showed that the gastric gland rudiment appeared at 17 dph. The basic structure of the stomach was fully developed between 26 and 35 dph. Two pepsinogen genes, named OpPGA1 and OpPGA2, were identified in the spotted knifejaw genome. qPCR results of developmental period showed that the two genes were low in expression during early development (5 and 15 dph). At 20 dph, the two genes started to show trace expression, and at 30 dph the mRNA expression levels of OpPGA1 and OpPGA2 reached the highest levels. Results of pepsin activity detection during the development period showed lower activity was detected 22 dph, followed by a peak at 30 dph. Under different feeding inductions, OpPGA1 showed the highest expression in the basic diet group and hard-shell group, while the expression level in the phytophagous group remained consistently low. The mRNA expression level of OpPGA2 in the phytophagous group was significantly higher than in other groups. Enzyme activity determination under different feeding inductions showed slightly higher enzyme activity in the basic diet group and crustacean group. The results of in situ hybridization showed that the mRNA of both OpPGA1 and OpPGA2 genes was both expressed in gastric gland cells. These information can contribute to the development of practical feeding methods in terms of digestive physiology for the development of larvae.

Antioxidant Status in the Blood of Psychosocially Stressed Rats Treated with Honey

Abstract Linden honey represents a unique honey variety valued for its nutritional benefits, distinctive taste and aroma. Phenols, polyphenols, flavonoids, ascorbic acid and phenolic acids in honey have antioxidant activities. This study aimed to investigate the effects of linden honey on the activities of antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPx), as well as on the concentration of malondialdehyde (MDA) in individually housed animals. The investigated parameters were quantified using spectrophotometric method for determination of enzyme activities and MDA concentration in the blood. We found that treatment with linden honey in the socially isolated animals significantly increased the enzyme activities of CAT and GPx, and significantly decreased the concentration of MDA. The modulation of CAT and GPx activities in socially isolated animals treated with linden honey may be very important for understanding the role of honey in the capacity of antioxidant defense system to increase and maintain its stability in psychosocial stress conditions. Our results may be important in biomedical research for understanding the role of honey in the amelioration of oxidative stress.

Effects of antioxidant alfa-lipolic acid on polyphenol oxidase activity of acitretin- methotrexate combination in rat spleen tissue

Objectives: Acitretin, a lipophilic weak acid, is a slightly water-soluble vitamin A analog. Methotrexate is a folic acid analogue and an antimetabolite used for the treatment of many diseases.. Alpha lipoic acid is a nutritional coenzyme with important physiological effects in protein, fat and carbohydrate metabolism. Polyphenol oxidase is an oxidoreductase containing copper in its active site and oxidizes phenolic compounds. The aim of this study is to determine the effect of Alpha lipoic acid on the polyphenol oxidase enzyme activity in the spleen tissue in removing the damage caused by the free radicals produced by Acitretin and Methotrexate at the cellular level. Materials and Methods: Study groups were formed as Control , Alpha lipoic acid , Acitretin+Methotrexate and Acitretin+Methotrexate+Alpha lipoic acid groups. Rats who were starved for 24 hours were injected at the every morning. Acitretin, Methotrexate and Alpha Lipoic Acid were dissolved in 0.9% NaCl and given to rats by intraperitoneal injection. Rats were sacrificed by cervical dislocation on the 7th day after injection. After perfusion of the heart, the spleen was removed. Extracted spleen tissue samples were used for polyphenol oxidase enzyme activity determination. Results: Results: Compared with groups Control; While 18% inhibition was observed in the Alpha lipoic acid group, 20% inhibition was observed in the Acitretin + Methotrexate group. When Alpha lipoic acid was added to the Acitretin + Methotrexate group, while 19% activation was observed according to Control, 1% activation was observed compared to the Acitretin+Methotrexate group. Although there were differences between the groups, these differences were not statistically significant. Conclusion: The inhibition of PPO enzyme activity by ALA can be explained by the fact that while ALA alone acts as a radical scavenger that can regenerate substances with antioxidant properties in the spleen tissue, when given in combination, it acts as an antioxidant that triggers PPO activity.