Crucial Enzyme Activities Research Articles

Directed evolution and metabolic engineering generate an Escherichia coli cell factory for de novo production of 4-hydroxymandelate

4-hydroxymandelate is a high-value aromatic compound used in the medicine, cosmetics, food, and chemical industry. However, existing natural extraction and chemical synthesis methods are costly and lead to environmental pollution. This study employed metabolic engineering and directed evolution strategies for de novo 4-hydroxymandelate biosynthesis. Two key challenges were addressed: insufficient precursor supply and limited activity of crucial enzymes. Through gene overexpression and multi-level gene interference using CRISPRi, An Escherichia coli chassis capable of producing the key precursor 4-hydroxyphenylpyruvate and the titer reached 5.05 mM (0.91 g/L). A mutant clone was obtained, HmaSV152G, which showed a 5.13-fold improvement in the catalytic rate. During fermentation, a high production of 194.87 mM (32.768 g/L) 4-hydroxymandelate was achieved in 76 h with a batch supply of glucose in a 5-L bioreactor. This study demonstrated the great potential of biosensors in protein engineering and provides a reference for large-scale production of other high-value aromatic compounds.

Comparative Genomic Analysis of the Mutant Rhodotorula mucilaginosa JH-R23 Provides Insight into the High-Yield Carotenoid Mechanism

In this study, the wild-type Rhodotorula mucilaginosa GDMCC 2.30 and its high carotenoid-producing mutant JH-R23, which was screened from the space mutation breeding treated wild type, were used as materials. Through whole-genome sequencing and resequencing analysis, the carotenoid metabolic pathway and mechanism of high carotenoid production in the mutant were explored. The R. mucilaginosa GDMCC 2.30 genome comprised 18 scaffolds and one circular mitochondrial genome with a total size of 20.31 Mb, a GC content of 60.52%, and encoding 7128 genes. The mitochondrial genome comprised 40,152 bp with a GC content of 40.59%. Based on functional annotations in the GO, KEGG, and other protein databases, nine candidate genes associated with carotenoid metabolic pathways, and candidate genes of the CrtS and CrtR homologous gene families were identified. The carotenoid metabolic pathway was inferred to start from sugar metabolism to the mevalonate pathway, as is common to most fungi, and the final product of the mevalonate pathway, geranylgeranyl diphosphate, is a precursor for various carotenoids, including β-carotene, lycopene, astaxanthin, and torularhodin, formed through the activity of crucial enzymes encoded by genes such as CrtI, CrtYB, CrtS, and CrtR. Resequencing analysis of the mutant JH-R23 detected mutations in the exons of four genes, including those encoding Gal83, 3-oxoacyl-reductase, p24 proteins, and GTPase. These mutations are interpreted to have an important impact on carotenoid synthesis by JH-R23.

Influence of different baking temperatures of red kojic rice on the physicochemical properties, antioxidant capacity, and functional components of red starter wine.

To improve the quality of red starter wine, this study explored the effects of baking red kojic rice at varying temperatures on the physicochemical characteristics of red starter wine. Baking was predicated on understanding crucial enzyme activities and starch granule structure of red kojic rice at 75, 95, and 105 °C, leading to the production of three red starter wine variants (BHQW1, BHQW2, and BHQW3). The results revealed an increased alcohol (increase 0.50%), total sugar (increase 0.14 g L-1 ), and total acid (increase 0.54 g L-1 ) content in red starter wine fermented using baked red kojic rice compared with the control group (wine fermented with unbaked rice, HQW). Furthermore, both the 105 °C baked red kojic rice and its resulting BHQW3 demonstrated significantly higher red color values than HQW (increase 2.03 U g-1 and 0.15 U mL-1 respectively). The highest lovastatin content was presented in red kojic rice baked at 105 °C and its corresponding fermented wine (1420.63 ± 507.9 μg g-1 and 3368.87 ± 228.16 μg L-1 respectively). Additionally, BHQW groups displayed higher total flavonoids and phenols content than HQW. Regarding antioxidant capacity, all BHQW groups showed stronger overall antioxidant capacity than HQW. The determination of volatile components revealed the highest content of volatile compounds in BHQW2 (2621.19 ± 548.24 μg L-1 ) and significantly higher volatile esters in BHQW1 (254.46 ± 16.63 μg L-1 ). Moreover, 16 volatile compounds were identified only in BHQW groups, including isoamyl caprylate, 2-ethylhexyl alcohol, and benzaldehyde. Our findings suggested that the baking technique of red kojic rice could enhance the quality of red starter wine through enhancing antioxidant properties, increasing functional components, and enriching volatile flavor compounds, thus providing a foundation for new techniques in red starter wine production. © 2023 Society of Chemical Industry.

Effects of acute hypoxic post-feeding condition on respiratory metabolism and digestion in yellow catfish (Pelteobagrus fulvidraco)

In this study, acute hypoxia was applied to two states of yellow catfish (Pelteobagrus fulvidraco) (non-feeding and feeding) for 48 h. We investigated the effects of acute hypoxia on the respiratory metabolism and digestive ability of yellow catfish (mean body length: 7.61 ± 0.52 cm). The results indicated that the respiratory frequency of yellow catfish under acute hypoxic post-feeding condition was rapidly accelerated and the oxygen consumption rate was reduced. The level of succinate dehydrogenase in the gills first increased and then significantly decreased (p < 0.05) and the level of lactate dehydrogenase significantly increased (p < 0.05). When the expression level of hypoxia-inducing factor was significantly increased (p < 0.05), the downstream coding genes (hexokinase 1 (hk1), phosphofructokinase (pfkla), and pyruvate kinase M1/2a (pkma)), key molecules of glycolysis, and the activity of crucial enzymes (hexokinase, phosphofructokinase, and pyruvate kinase) increased. At the same time, the glucose level dropped and the lactic acid level increased, indicating that the metabolic mode in the gills of yellow catfish under acute hypoxic post-feeding condition gradually changed to anaerobic, and the anaerobic glycolysis process was activated to provide the required energy for the body. In addition, we found that the activity of digestive enzymes (trypsin, lipase, and amylase) and expression levels of digestive genes (trypsin, lipase, and amylase) were inhibited in the liver and intestine of yellow catfish under the acute hypoxic post-feeding condition, indicating that their digestive ability was reduced. This may adversely affect growth and development. This study analyzed the effects of acute hypoxic post-feeding exposure on the respiratory metabolism and digestive capacity of yellow catfish, providing new insights into fish physiology and sustainable fish culture.

Natural products modulating epigenetic mechanisms by affecting histone methylation/demethylation: targeting cancer cells.

Many natural products can exert anticancer or chemopreventive activity by interfering with the cellular epigenetic machinery. While a plethora of studies indicate the relevance of affecting DNA methylation and histone acetylation, the influence on the mechanisms related to histone methylation is often overlooked. This may be associated with the lagging evidence that changes in the action of histone methylation writers and erasers and subsequent alterations in the profile of histone methylation are causally related with carcinogenesis. However, recent animal studies have shown that targeting histone methylation/demethylation affects the course of experimentally induced carcinogenesis. Existing data suggest that numerous natural compounds from different chemical groups, including green tea polyphenols and other flavonoids, curcuminoids, stilbene derivatives, phenolic acids, isothiocyanates, alkaloids and terpenes, can affect the expression and activity of crucial enzymes involved in the methylation and demethylation of histone lysine and arginine residues. These activities have been associated with the modulation of cancer-related gene expression and functional changes, including reduced cell proliferation and migration, and enhanced apoptosis in various cancer models. Most studies focused on the modulation of the expression and/or activity of two proteins - EZH2 (a H3K27 methyltransferase) and LSD1 (a H3K4/9 demethylase), or the effects on the global levels of histone methylation marks by the phytochemicals, but data regarding other histone methyltransferases or demethylases are scarce. While the field still remains relatively unexplored, this review aims to explore the impact of natural products on the enzymes related to histone methylation/demethylation, showing their relevance to carcinogenesis and cancer progression.

Initial Study of Fungal Bioconversion of guishe (Agave lechuguilla Residue) Juice for Bioherbicide Activity on Model Seeds

In agriculture, weed management is a significant concern because their uncontrolled proliferation decreases soil quality for food crops. Allelopathy is a natural phenomenon in which the activity of allelochemical compounds inhibits the germination and growth of invasive plants as a defense mechanism. Among allelochemicals are polyphenols, which may affect genetic material or crucial enzyme activities for proper physiological function. Agroindustrial residues are a vast source of polyphenolic compounds with allelochemical activity. The bagasse of Agave Lechuguilla, known as guishe, is an abundant residue in México. The guishe has been characterized before by its polyphenolic content. Based on that, a fungal bioconversion process was developed to increase the availability of the allelochemicals in the guishe juice. First, guishe juice was obtained by mechanical pressed and characterized by spectrophotometric analysis. Results showed (g/L): 5.62 flavonoids, 0.64 of hydrolyzable polyphenols, 12.67 of reducing sugars, and 23.3 total sugars. The compounds detected by HPLC-ESI-MS were pterostilbene, hydroxycaffeic, caffeoyltartaric, and 4-O-glucoside coumaric acids, considered allelopathic. After the fungal bioprocess, (+)-gallocatechin and 3,7-Dimethyl quercetin were detected as additional compounds of interest. The flavonoid and hydrolyzable polyphenol content were modified to the highest accumulation of 1.57 and 14.9 g/L at 72 h, meaning a 2.45- and 2.22-fold increase. A bioprocess guishe juice (BGJ) was obtained at the compound accumulation peak of 72 h and evaluated in an allelopathic assay on model seeds (tomato and corn). Results show that BGJ inhibits up to 96.67% of corn seeds and up to 76.6% of tomato seeds compared to positive control.

Citrus aurantium ‘Crispifolia’ Essential Oil: A Promise for Nutraceutical Applications

Food waste is one of the main topics of various scientific studies of the last decade. In this regard, this work analyzed an essential oil (EO) extracted from the flavedo of Citrus aurantium ‘Crispifolia’ fruit. The analysis, performed by GC-MS, showed a chemically variegated chromatogram characterized by the presence of limonene (33.35%), but also by oxygenated monoterpenes such as β-linalool (7.69%), α-terpineol (7.06%), and geranyl acetate (10.12%). EO from the external part of the C. aurantium peel had several properties, including excellent antimicrobial and good antibiofilm activities. It also showed antioxidant activity in vitro and decreased the amount of cellular ROS, thus stimulating the catalytic activity of crucial enzymes involved in mitigating oxidative stress.

Influences of flavonoids from Sedum aizoon L. on the cell membrane of Botrytis cinerea

Botrytis cinerea is a plant pathogen, which affects the quality of fruits. However, chemical antifungal agents are extremely harmful to the environment, hence it's critical to create a natural plant-derived antifungal agent to replace chemical antifungal agents. The study explored the antifungal mechanisms of flavonoids from Sedum aizoon L. (FSAL) against B. cinerea. In the study, cell membrane served as an important breakthrough point. The minimum inhibitory concentration (MIC) of FSAL against B. cinerea was investigated. The propidium iodide (PI) staining observation of B. cinerea was explored. The activities of crucial enzymes involved in the metabolism were studied as well as the phospholipid and fatty acid changes. The expression of genes related to the membrane lipid metabolism was also measured. The results showed that FSAL had a certain inhibitory effect on B. cinerea and the MIC was 1.500 mg/mL. FSAL disrupted the cell membrane of B. cinerea, which decreased the contents of glutathione and proline at a low level as well as the membrane phospholipids. Compared with the control group, the activities of crucial enzymes were decreased, and the expression of key genes was reduced. It indicated that FSAL inhibited B. cinerea by disrupting its cell membrane, offering the possibility of a natural plant-derived antifungal agent for the preservation of fruits.

Rice spikelet formation inhibition caused by decreased sugar utilization under high temperature is associated with brassinolide decomposition

Rice (Oryza sativa) spikelet formation is dependent on carbohydrate metabolism. High temperature inhibits young panicle growth and spikelet formation, and brassinolides (BRs) are involved in the regulation of spikelet formation. Elucidation of the mechanism by which endogenous BRs mediate carbohydrate metabolism to influence spikelet formation under high temperature may provide a theoretical and technical basis to alleviate heat injury. In this study, rice cultivar ‘IR36’ was subjected to high temperature (HT; 40 °C) or normal temperature (NT; 33 °C) for 7 days at the panicle initiation stage in conjunction with application of exogenous 2, 4-epibrassinolide (EBR) and brassinazole (BRZ) at the beginning of the treatments. HT inhibited spikelet differentiation and aggravated spikelet degeneration to reduce the number of spikelets per panicle. Exogenous EBR reduced the decrease in spikelet number induced by HT, whereas exogenous BRZ enhanced the reduction in spikelet number. Analyses of the transcript levels of genes associated with BR biosynthesis and decomposition showed that HT promoted BR decomposition to reduce the endogenous BR content. HT blocked sucrose transport from leaves to young panicles, and inhibited the activities of crucial enzymes involved in sucrose hydrolysis, glycolysis, and the tricarboxylic acid cycle to reduce sugar utilization. In addition, HT reduced the activities of antioxidant enzymes and increased the contents of hydrogen peroxide and malondialdehyde. Exogenous EBR application promoted sucrose transport to young panicles and improved sucrose utilization under NT and HT, whereas application of BRZ resulted in the opposite physiological response. These results show that inhibition of spikelet formation owing to decreased sugar utilization under HT is associated with BR decomposition.

Extracts from Almond (Terminalia catappa) leaf and stem bark mitigate the activities of crucial enzymes and oxidative stress associated with hypertension in cyclosporine A-stressed rats.

We investigated the effect of extracts from the leaf (ALE) and stem bark (ABE) of Almond tree on activities of some crucial enzymes [angiotensin-1 converting enzyme (ACE), arginase, acetylcholinesterase (AChE), phosphodiesterase-5 (PDE-5), adenosine deaminase (ADA), superoxide dismutase (SOD), catalase], and thiobarbituric acid reactive species (TBARS) associated with hypertension in normal adult male Wistar albino rats and Cyclosporine A (CsA)-stressed rats. The result revealed that CsA-stressed rats treated with captopril and extracts (ALE and ABE) had lowered ACE, arginase, AChE, PDE-5, ADA activities, and TBARS level, coupled with improved SOD and catalase activities compared with untreated CsA-stressed rats, which had reversed these biochemicals compared to normal rats. This suggests that the extracts could be explored to suppress hypertension and other cardiac injury known with CsA treatment; the potentials that could be linked with the constituent polyphenols. However, further studies including blood pressure should be determined to ascertain this claim. PRACTICAL APPLICATIONS: Drug-induced cardiotoxicity, hypertension, and organ damage are among the most common side effects of pharmaceutics. Therefore, it becomes imperative to find natural, effective, and alternative therapy with little or no side effect to combat drug toxicity. The use of Almond (leaf and stem bark) in folklore for the treatment/management of hypertension and other heart-related diseases without full scientific basis is on the increase. Hence, this study provides some biochemical evidences on the effect of Almond leaf and stem back extracts on crucial enzymes and oxidative stress markers involve in the incidence of hypertension in the course of Cyclosporine A administration. The findings of this study indicated that the studied plant materials could be promoted as nutraceutical agents to neutralize drug-induced cardiac injury and hypertension.

Inhibition to crucial enzymes in the lethal effects of the dinoflagellate Karenia mikimotoi on the rotifer Brachionus plicatilis

Blooms of the dinoflagellate Karenia mikimotoi have cause great financial losses to the marine aquaculture industry. However, the toxicity mechanism of this species is still not fully known. In this study, we evaluated the short-term effects of K. mikimotoi on the rotifer Brachionus plicatilis by micro and sub micro observing and by measuring inhibition of crucial enzymes. Behaviour disorder, mucus production, corona and cilium damage, vesical production, and body shrinkage occurred within 1 h after rotifers were treated with K. mikimotoi at a density of 3 × 104 cells/mL. Enzyme activity assays showed that K. mikimotoi at low densities significantly inhibited multiple enzymes within 3 h, and obvious density-effect trends were also observed. For instance, activity of esterase and acetylcholinesterase of rotifers significantly decreased to 94.3/83.3% and 82.8/66.9% of control treatment values in 30 and 1000 cells/mL algal treatment, respectively. Total ATPase and Na+-K+-ATPase activities of rotifers also decreased to 82.3% and 68.6% of control values in 1000 cells/mL treatment. The LDH releasement test and MDA tests showed no significant difference between algae treatment and control. It suggested that K. mikimotoi might not cause significant cytolysis and oxidative damage to rotifers, but may cause mortality by inhibiting the activity of crucial enzymes, which may lead to cell permeability disorder and body shrinkage.

Effects of gestational thiamine-deprivation and/or exposure to ethanol on crucial offspring rat brain enzyme activities

Objective The fetal alcohol spectrum disorder (FASD) is a group of clinical conditions associated with the in utero exposure to ethanol (EtOH). We have recently examined the effects of a moderate maternal exposure to EtOH on crucial brain enzyme activities in offspring rats, and discussed the translational challenges arising when attempting to simulate any of the clinical conditions associated with FASD. Materials and methods In this current study, we: (i) address the need for a more consistent and reliable in vivo experimental platform that could simulate milder cases of FASD complicated by simultaneous thiamine-deprivation during gestation and (ii) explore the effects of such a moderate maternal exposure pattern to EtOH and a thiamine-deficient diet (TDD) on crucial enzyme activities in the offspring rat brains. Results We demonstrate a significant decrease in the newborn and 21-day-old offspring body and brain weight due to maternal dietary thiamine-deprivation, as well as evidence of crucial brain enzyme activity alterations that in some cases are present in the offspring rat brains long after birth (and the end of the maternal exposure to both EtOH and TDD). Conclusions Our findings provide a preliminary characterization of important neurochemical effects due to maternal exposure to EtOH and TDD during gestation that might affect the offspring rat neurodevelopment, and that characterization should be further explored in a brain region-specific manner level as well as through the parallel examination of changes in the offspring rat brain lipid composition.

Cold plasma treatment induces phenolic accumulation and enhances antioxidant activity in fresh-cut pitaya (Hylocereus undatus) fruit

Non-thermal cold plasma as a novel method for maintaining safety and inducing phenolic accumulation in fresh-cut pitaya (Hylocereus undatus) fruit and the possible mechanisms were investigated. Results showed that cold plasma treatment (60 kV for 5 min) significantly inhibited the growth of total aerobic bacterial counts, increased the cutting-induced phenolic accumulation and enhanced antioxidant activity in fresh-cut pitaya fruit. Moreover, cold plasma treatment promoted the consumption of main sugars, enhanced the level of energy status, accelerated the generation of reactive oxygen species (ROS) at earlier storage, increased gene expression and activity of crucial enzymes in phenylpropanoid pathway. The present research provides convincing evidence regarding the beneficial effects of cold plasma treatment for inducing phenolic accumulation and enhancing antioxidant activity of fresh-cut pitaya fruit and offers potential opportunity for the processing and preservation of fresh-cut fruits and vegetables.

Oxidative stress, apoptosis activation and symbiosis disruption in giant clam Tridacna crocea under high temperature

Giant clams are one of the most important animals in coral reef ecosystem, and its growth and reproduction are being threatened by heat stress due to global warming. In the present study, the symbiont density, the crucial enzyme activities and the transcriptome were investigated in the outer mantle of giant clam Tridacna crocea after the acute exposure of high temperature. The density of symbiotic zooxanthellae decreased significantly during 12–24 h, with the minimum level (7.75 × 105 cell cm−2, p < 0.05) at 12 h after heat stress. The activities of superoxide dismutase in the heat stress group was significantly lower than that in the control group at 24 h after heat stress, while no significant change in the activities of catalase was observed during the entire stress process. The activation level of caspase3 began to increase significantly at 12 h (1.22-fold, p < 0.05), and reached the highest level at 24 h (1.38-fold, p < 0.05) after heat stress. Six paired-end libraries were sequenced in two groups, including the heat stress and control group at 12 h after heat stress. Through the assembling of 187,116,632 paired-end reads with lengths of 2 × 150 bp, a total of 26,676 genes were obtained which derived from giant clam. Bioinformatics analysis revealed 47 significantly upregulated and 88 significantly downregulated genes at 12 h after the treatment. There were 12 overrepresented GO terms for significantly upregulated genes, mostly related to unfolded protein binding and ATP binding, whereas no GO term was overrepresented for significantly downregulated genes. These results collectively suggest high temperature could induce excessive oxidative stress through the repressed antioxidant ability, the apoptosis activated by the unfolded protein response, and further the collapse of the symbiosis between host and symbiont, which has been threatening the growth and reproduction of the giant clam T. crocea.

The influence of chosen fungicides on the activity of aminopeptidases in winter oilseed rape during pods development

Cultivation of oilseed rape requires application of specific fungicides. Besides their protective role, they can potentially influence the expression and activity of crucial enzymes in the plant. Among the large number of enzymes expressed in plants, aminopeptidases play a key role in all crucial physiological processes during the whole life cycle (e.g. storage protein mobilization and thus supplying plant with needed amino acids, as well as plant aging, protection and defense responses). In the present paper, we evaluate for the first time, the influence of the treatment of winter oilseed rape with commercially available fungicides (Pictor 400 SC, Propulse 250 SE and Symetra 325 SC), on the activity of aminopeptidases expressed in each plant organ (flowers, leaves, stems and pods separately). Fungicides were applied once, at one of the three stages of oilseed rape development (BBCH 59–61, BBCH 63–65 and BBCH 67–69). The aminopeptidase activity was determined using six different amino acid p-nitroanilides as substrates. The results have shown, that in control plants, at the beginning of intensive pods development and seeds production, hydrophobic amino acids with bulky side chains (Phe, Leu) were preferentially hydrolysed. In control plants, the activity was ~3.5 times higher in stems and pods, compared to leaves. The treatment with all pesticides caused significant increase in aminopeptidases hydrolytic activity toward small amino acids Gly, Ala as well as proline, mostly in flowers and leaves. These amino acids are proven to be crucial in the mechanisms of delaying of plant aging, development of better resistance to stress and plant defense. It can be suggested, that studied fungicides enhance such mechanisms, by activating the expression of genes coding for aminopeptidases, which are active in hydrolysis of N-terminal amino acids such as Gly, Ala, Pro from storage peptides and proteins. Depending on fungicide, the major increase of aminopeptidase activity was observed after application at BBCH 67–69 (Pictor 400 SC and Symetra 325 SC) and BBCH 63–65 (Propulse 250 SE) stages of development. Our study revealed, that agrochemical treatment and time of application, influenced the expression and activity of aminopeptidases, even though they were not molecular targets of applied fungicides. Since aminopeptidases are widely distributed throughout all organisms and are crucial in many key physiological processes, it can be expected, that factors influencing their expression and activity in plants, can also influence these enzymes in other organisms, especially humans and other mammals.

Reconstruction of metabolic module with improved promoter strength increases the productivity of 2-phenylethanol in Saccharomyces cerevisiae

Background2-phenylethanol (2-PE) is an important aromatic compound with a lovely rose-like scent. Saccharomyces cerevisiae is a desirable microbe for 2-PE production but its natural yield is not high, and one or two crucial genes’ over-expression in S. cerevisiae did not improve 2-PE greatly.ResultsA new metabolic module was established here, in which, permease Gap1p for l-phenylalanine transportation, catalytic enzymes Aro8p, Aro10p and Adh2p in Ehrlich pathway respectively responsible for transamination, decarboxylation and reduction were assembled, besides, glutamate dehydrogenase Gdh2p was harbored for re-supplying another substrate 2-oxoglutarate, relieving product glutamate repression and regenerating cofactor NADH. Due to different promoter strengths, GAP1, ARO8, ARO9, ARO10, ADH2 and GDH2 in the new modularized YS58(G1-A8-A10-A2)-GDH strain enhanced 11.6-, 15.4-, 3.6-, 17.7-, 12.4- and 7.5-folds respectively, and crucial enzyme activities of aromatic aminotransferases and phenylpyruvate decarboxylase were 4.8- and 7-folds respectively higher than that of the control.ConclusionsUnder the optimum medium and cell density, YS58(G1-A8-A10-A2)-GDH presented efficient 2-PE synthesis ability with ~ 6.3 g L−1 of 2-PE titer in 5-L fermenter reaching 95% of conversation ratio. Under fed-batch fermentation, 2-PE productivity at 24 h increased 29% than that of single-batch fermentation. Metabolic modularization with promoter strategy provides a new prospective for efficient 2-PE production.

Molybdenum and iron mutually impact their homeostasis in cucumber (Cucumis sativus) plants.

Molybdenum (Mo) and iron (Fe) are essential micronutrients required for crucial enzyme activities in plant metabolism. Here we investigated the existence of a mutual control of Mo and Fe homeostasis in cucumber (Cucumis sativus). Plants were grown under single or combined Mo and Fe starvation. Physiological parameters were measured, the ionomes of tissues and the ionomes and proteomes of root mitochondria were profiled, and the activities of molybdo-enzymes and the synthesis of molybdenum cofactor (Moco) were evaluated. Fe and Mo were found to affect each other's total uptake and distribution within tissues and at the mitochondrial level, with Fe nutritional status dominating over Mo homeostasis and affecting Mo availability for molybdo-enzymes in the form of Moco. Fe starvation triggered Moco biosynthesis and affected the molybdo-enzymes, with its main impact on nitrate reductase and xanthine dehydrogenase, both being involved in nitrogen assimilation and mobilization, and on the mitochondrial amidoxime reducing component. These results, together with the identification of >100 proteins differentially expressed in root mitochondria, highlight the central role of mitochondria in the coordination of Fe and Mo homeostasis and allow us to propose the first model of the molecular interactions connecting Mo and Fe homeostasis.

Experimentally-induced maternal hypothyroidism alters crucial enzyme activities in the frontal cortex and hippocampus of the offspring rat

Thyroid hormone insufficiency during neurodevelopment can result into significant structural and functional changes within the developing central nervous system (CNS), and is associated with the establishment of serious cognitive impairment and neuropsychiatric symptomatology. The aim of the present study was to shed more light on the effects of gestational and/or lactational maternal exposure to propylthiouracil (PTU)-induced hypothyroidism as a multilevel experimental approach to the study of hypothyroidism-induced changes on crucial brain enzyme activities of 21-day-old Wistar rat offspring in a brain region-specific manner. This experimental approach has been recently developed and characterized by the authors based on neurochemical analyses performed on newborn and 21-day-old rat offspring whole brain homogenates; as a continuum to this effort, the current study focused on two CNS regions of major significance for cognitive development: the frontal cortex and the hippocampus. Maternal exposure to PTU in the drinking water during gestation and/or lactation resulted into changes in the activities of acetylcholinesterase and two important adenosinetriphosphatases (Na(+),K(+)- and Mg(2+)-ATPase), that seemed to take place in a CNS-region-specific manner and that were dependent upon the PTU-exposure timeframe followed. As these findings are analyzed and compared to the available literature, they: (i) highlight the variability involved in the changes of the aforementioned enzymatic parameters in the studied CNS regions (attributed to both the different neuroanatomical composition and the thyroid-hormone-dependent neurodevelopmental growth/differentiation patterns of the latter), (ii) reveal important information with regards to the neurochemical mechanisms that could be involved in the way clinical hypothyroidism could affect optimal neurodevelopment and, ultimately, cognitive function, as well as (iii) underline the need for the adoption of more consistent approaches towards the experimental simulation of congenital and early-age-occurring hypothyroidism.

Raffinose in seedlings of winter vetch (Vicia villosa Roth.) under osmotic stress and followed by recovery

During germination of winter vetch (Vicia villosa Roth.) seeds, the degradation of raffinose family oligosaccharides and galactosyl pinitols occurred faster in axis than in cotyledons. After 7 days of germination, all α-d-galactosides disappeared and the soluble carbohydrates in seedling tissues consisted of d-pinitol, sucrose, fructose, glucose and myo-inositol. Osmotic stress caused by incubation of seedlings in PEG 8000 solution (−0.5, −1.0, and −1.5 MPa) for 48 h induced the activity of crucial enzymes of the RFOs pathway, i.e. galactinol synthase and raffinose synthase, in both the root and epicotyl but not in cotyledons. The root and epicotyl accumulated elevated amounts of galactinol and raffinose as the osmotic potential was lowered. This process was transient because when PEG solution was replaced with water, galactinol and raffinose were degraded, thus confirming their direct involvement in the response of tissues to osmotic stress. Among other soluble carbohydrates, only sucrose accumulated in response to stress. The results did not show potential role of d-pinitol in the adjustment of winter vetch seedlings to osmotic stress.

Choline-deprivation alters crucial brain enzyme activities in a rat model of diabetic encephalopathy

Diabetic encephalopathy describes the moderate cognitive deficits, neurophysiological and structural central nervous system changes associated with untreated diabetes. It involves neurotoxic effects such as the generation of oxidative stress, the enhanced formation of advanced glycation end-products, as well as the disturbance of calcium homeostasis. Due to the direct connection of choline (Ch) with acetylcholine availability and signal transduction, a background of Ch-deficiency might be unfavorable for the pathology and subsequently for the treatment of several metabolic brain diseases, including that of diabetic encephalopathy. The aim of this study was to shed more light on the effects of adult-onset streptozotocin (STZ)-induced diabetes and/or Ch-deprivation on the activities of acetylcholinesterase (AChE) and two important adenosine triphosphatases, namely Na(+),K(+)-ATPase and Mg(2+)-ATPase. Male adult Wistar rats were divided into four main groups, as follows: control (C), diabetic (D), Ch-deprived (CD), and Ch-deprived diabetic (D+CD). Deprivation of Ch was provoked through the administration of Ch-deficient diet. Both the induction of diabetes and the beginning of dietary-mediated provoking of Ch-deprivation occurred at the same day, and rats were killed by decapitation after 30 days (1 month; groups C1, D1, CD1 and D1+CD1) and 60 days (2 months; groups C2, D2, CD2 and D2+CD2, respectively). The adult rat brain AChE activity was found to be significantly increased by both diabetes (+10%, p < 0.001 and +11%, p < 0.01) and Ch-deprivation (+19%, p < 0.001 and +14%, p < 0.001) when compared to the control group by the end of the first (C1) and the second month (C2), respectively. However, the Ch-deprived diabetic rats' brain AChE activity was significantly altered only after a 60-day period of exposure, resulting in a +27% increase (D2+CD2 vs. C2, p < 0.001). Although the only significant change recorded in the brain Na(+),K(+)-ATPase activity after the end of the first month is attributed to Ch-deprivation (+21%, p < 0.05, CD1 vs. C1), all groups of the second month exhibited a statistically significant decrease in brain Na(+),K(+)-ATPase activity (-24%, p < 0.01, D2 vs. C2; -21%, p < 0.01, CD2 vs. C2; -22%, p < 0.01, D2+CD2 vs. C2). As concerns Mg(2+)-ATPase, the enzyme's activity demonstrates no significant changes, with the sole exception of the D2+CD2 group (+21%, p < 0.05, D2+CD2 vs. C2). In addition, statistically significant time-dependent changes concerning the brain Mg(2+)-ATPase activity were recorded within the diabetic (p < 0.05, D2 vs. D1) and the Ch-deprived (p < 0.05, CD2 vs. CD1) rat groups. Our data indicate that Ch-deprivation seems to be an undesirable background for the above-mentioned enzymatic activities under untreated diabetes, in a time-evolving way. Further studies on the issue should focus on a region-specific reevaluation of these crucial enzymes' activities as well as on the possible oxidative mechanisms involved.