Biosynthesis Enzyme Activity Research Articles

Effects of high temperature on grain quality and enzyme activity in heat-sensitive versus heat-tolerant rice cultivars.

High-temperature (HT) stress significantly affects the quality of rice (Oryza sativa L.), although the underlying the mechanism remains unknown. Therefore, in the present study, we assessed protein components, amino acids, mineral element levels, starch biosynthesis enzyme activity and gene expression of two heat-sensitive and two heat-tolerant genotypes under HT treatment during early (from 1 to 10 days, T1) and mid-filling (from 11 to 20 days, T2) after anthesis. Except for one cultivar, most rice varieties exhibited increased levels of amylose, chalky degree and protein content, along with elevated cracked grains and pasting temperatures and, consequently, suppressed amino acid levels under HT stress. HT treatment also increased protein components, macro- (Mg, K, P and S) and microelements (Cu, Zn, and Mo) in the rice flour. Both HT treatments reduced the activity of ADP-glucose pyrophosphate, ground-bound starch synthase, as well as the relative ratio of amylose to total starch, at the same time increasing starch branch enzyme activity. The expression levels of OsAGPL2, OsSSS1 and OsSBE1 in all varieties exhibited the same trends as enzyme activity under HT treatment. High temperatures negatively affected rice quality during grain filling, which is related to heat tolerance and grain shape. Altered enzymatic activity is crucial to compensate for the lowered enzyme quality under heat stress. © 2024 Society of Chemical Industry.

Preparation and Characterization of Heparan Sulfate-Derived Oligosaccharides to Investigate Protein-GAG Interaction and HS Biosynthesis Enzyme Activity.

Heparan sulfate chains are complex and structurally diverse polysaccharides that interact with a large number of proteins, thereby regulating a vast array of biological functions. Understanding this activity requires obtaining oligosaccharides of defined structures. Here we describe methods for isolating, engineering, and characterizing heparan sulfate-derived oligosaccharides and approaches based on high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and bio-layer interferometry (BLI) to study their structures, modifications, and interactions.

Adaptation to the coupling of glycolysis to toxic methylglyoxal production in tpiA deletion strains of Escherichia coli requires synchronized and counterintuitive genetic changes

Methylglyoxal is a highly toxic metabolite that can be produced in all living organisms. Methylglyoxal was artificially elevated by removal of the tpiA gene from a growth optimized Escherichia coli strain. The initial response to elevated methylglyoxal and its toxicity was characterized, and detoxification mechanisms were studied using adaptive laboratory evolution. We found that: 1) Multi-omics analysis revealed biological consequences of methylglyoxal toxicity, which included attack on macromolecules including DNA and RNA and perturbation of nucleotide levels; 2) Counter-intuitive cross-talk between carbon starvation and inorganic phosphate signalling was revealed in the tpiA deletion strain that required mutations in inorganic phosphate signalling mechanisms to alleviate; and 3) The split flux through lower glycolysis depleted glycolytic intermediates requiring a host of synchronized and coordinated mutations in non-intuitive network locations in order to re-adjust the metabolic flux map to achieve optimal growth. Such mutations included a systematic inactivation of the Phosphotransferase System (PTS) and alterations in cell wall biosynthesis enzyme activity. This study demonstrated that deletion of major metabolic genes followed by ALE was a productive approach to gain novel insight into the systems biology underlying optimal phenotypic states.

Grain density and its impact on grain filling characteristic of rice: mechanistic testing of the concept in genetically related cultivars

Physiological factors controlling assimilate partitioning was compared in relation to panicle architecture of lax- (Upahar) and compact-panicle (Mahalaxmi) rice cultivars. Grain number and ethylene production at anthesis are low, but filling rate is high in the former compared to high grain number and ethylene production and poor filling trait of the latter. Similar to Mahalaxmi, its progenitors Pankaj and Mahshuri, had attributes of high grain number and grain density, but grain filling was higher and ethylene evolution was lower. Disturbed genetic coherence owing to imbalance of gene groups brought in the cross combinations of Mahshuri and Pankaj could be responsible for high ethylene production leading to semi sterility of Mahalaxmi as the hormone slackened endosperm starch bio-synthesis enzyme activities. Mahalaxmi inherited grain compactness trait of its progenitors, but not the physiological attribute for reduced ethylene production, which impacted grain filling. Upahar, the progeny genotype of Mahalaxmi and IR62 cross, inherited the dominant allele for low ethylene production and good grain filling traits from the high yielding IR62. In conclusion grain filling in compact-panicle rice becomes poor subject to expression of recessive allele for high ethylene production, but the allele is amenable for suppression by corresponding dominant allele in a genetic breeding.

Intensification of surfactants’ synthesis by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8 on fried oil and glycerol containing medium

Searching for the ways to process waste has become very topical today. Biotechnology is one of the most environmentally attractive methods, which has the ability to solve the problem of waste utilization and to produce the valuable microbial products, for example biosurfactants. We concluded that fried sunflower oil, oil-containing wastes (soapstock) and glycerol can be used for biosurfactant production by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8. Glucose addition (0.1%) into the medium with fried oil (2vol.%) led to a 4-fold increase of final surfactant concentration (6.8g/L). The simultaneous addition of fumarate and citrate (0.01–0.2%) into the IMV B-7241 and K-8 strains’ cultivating medium was accompanied by an increase of the exocellular biosurfactant quantity by 2–2.5-fold compared to the cultivation without organic acids. An increase in surfactant concentration of IMV B-7241 strain was the result of the simultaneous functioning of two anaplerotic pathways, also resulting in a 3–5-fold increase in activity of biosynthesis enzymes. Cultivating on a mixture of glycerol and n-hexadecane (0.5–1.0vol.%) led to a 1.5–3-fold increasing surfactant synthesis. Biosurfactant preparations of IMV B-7241 (0.15–0.22mg/mL) and IMV Ac-5017 (0.61–2.1mg/mL) strains were effective against Escherichia coli IEM-1 (67% of cell loss), and vegetative (45–100% of cell loss) and spore (75% of spore loss) cells of Bacillus subtilis BT-2.

Antioxidative Activity of Amino Acids on Tissue Oxidative Stress in Human Intestinal Epithelial Cell Model

The protective effects of amino acids against H 2O 2-induced oxidative stress were investigated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were pretreated with amino acids (1, 2, and 5 mM) for 2 h and then stimulated with 1 mM H 2O 2 for 6 h. The secretion of IL-8, a proinflammatory mediator, was determined by ELISA as an indicator of tissue oxidative stress. The inhibition of H 2O 2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment with Cys, Val, Ile, Leu, Trp, His, Lys, and Ala. Cys enhanced glutathione (GSH) biosynthesis enzyme activity and increased cellular GSH levels. Branched-chain amino acids such as Val, Ile, and Leu elevated activities of GSH S-transferase (GST) and catalase. Trp, His, and Lys caused increases in GST activity. Ala enhanced GSH reductase activity. These data suggest that specific amino acids exert protective effects against tissue oxidative stress in intestinal epithelial cells based on the structure.

PPARα-activation results in enhanced carnitine biosynthesis and OCTN2-mediated hepatic carnitine accumulation

In fasted rodents hepatic carnitine concentration increases considerably which is not observed in PPARα−/− mice, indicating that PPARα is involved in carnitine homeostasis. To investigate the mechanisms underlying the PPARα-dependent hepatic carnitine accumulation we measured carnitine biosynthesis enzyme activities, levels of carnitine biosynthesis intermediates, acyl-carnitines and OCTN2 mRNA levels in tissues of untreated, fasted or Wy-14643-treated wild type and PPARα−/− mice. Here we show that both enhancement of carnitine biosynthesis (due to increased γ-butyrobetaine dioxygenase activity), extra-hepatic γ-butyrobetaine synthesis and increased hepatic carnitine import (OCTN2 expression) contributes to the increased hepatic carnitine levels after fasting and that these processes are PPARα-dependent.

Measurement of carnitine biosynthesis enzyme activities by tandem mass spectrometry: Differences between the mouse and the rat

Although the mouse frequently is used to study metabolism and deficiencies therein, little is known about carnitine biosynthesis in this animal. To this point, only laborious procedures have been described to measure the activity of carnitine biosynthesis enzymes using subcellular fractions as the enzyme source. We developed two simple tandem mass spectrometry-based methods to determine the activity of three carnitine biosynthesis enzymes (6- N-trimethyllysine dioxygenase, 4-trimethylaminobutyraldehyde dehydrogenase, and 4-trimethylaminobutyric acid dioxygenase) in total homogenates that can be prepared from frozen tissue. The new assays were used to characterize these enzymes in mouse liver homogenate. Because carnitine biosynthesis has been studied extensively in the rat, we compared the mouse tissue distribution of carnitine biosynthesis enzyme activities and levels of the biosynthesis metabolites with those in the rat to determine which tissues contribute to carnitine biosynthesis in these species. Surprisingly, large differences in enzyme activities were found between the rat and the mouse, whereas carnitine biosynthesis metabolite levels were very similar in both species, possibly due to the different kinetic properties of the first enzyme of carnitine biosynthesis. Also, muscle carnitine levels were found to vary considerably between these two species, suggesting that there is a metabolic dissimilarity between the mouse and the rat.

Co-ordinate regulation of sterol biosynthesis enzyme activity during accumulation of sterols in developing rape and tobacco seed

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol methyl transferase 1 and sterol acyltransferase, key enzymes involved in phytosterol biosynthesis were shown to be co-ordinately regulated during oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) seed development. In both plants, enzyme activities were low during the initial stages of seed development, increasing towards mid-maturation where they remained stable for a time, before declining rapidly as the oilseeds reached maturity. During seed development, the level of total sterols increased 12-fold in tobacco and 9-fold in rape, primarily due to an increase in steryl ester production. In both seed tissues, stages of maximum enzyme activity coincided with periods of high rates of sterol production, indicating developmental regulation of the enzymes to be responsible for the increases in the sterol content observed during seed development. Consistent with previous studies the data presented suggest that sterol biosynthesis is regulated by two key steps, although there may be others. The first is the regulation of carbon flux into the isoprenoid pathway to cycloartenol. The second is the flux from cycloartenol to Delta(5)-end-product sterols. The implications of the results in terms of enhancing seed sterol levels by genetic modification are also discussed.

P-70 - Effects of GABAergic drugs on cholecystokinin-like immunoreactivity and its biosynthesis enzyme activities in rat brain regions

P-70 - Effects of GABAergic drugs on cholecystokinin-like immunoreactivity and its biosynthesis enzyme activities in rat brain regions

Cytoprotective effect of reduced glutathione in hydrogen peroxide-induced endothelial cell injury

The kinetic effects of hydrogen peroxide (H 2O 2) on cultured endothelial cells isolated from bovine carotid artery were studied. The cytoprotective effects of glutathione (GSH) on H 2O 2-induced cell injury were also investigated. H 2O 2-induced a dose- and time-dependent cell injury in cultured endothelial cells. H 2O 2-induced cell injury was blocked by simultaneous treatment by catalase, but not by superoxide dismutase. H 2O 2 also induced endogenous PGI 2 biosynthesis, and the maximum PGI 2 production was reached after 1 h treatment. Stimulation of PGI 2 production was parallel with arachidonate release from H 2O 2-treated cells. However the prostaglandin biosynthesis enzyme activity in cells was inhibited by H 2O 2 treatment. When the cells were treated with GSH, the intracellular GSH reached a plateau after 3 h treatment. Both H 2O 2-induced cell injury and PGI 2 production were significantly inhibited by the 3 h pretreatment with GSH. The cytoprotective effect of GSH was completely inhibited by buthionine sulfoximine which is a specific inhibitor of γ-glutamylcysteine synthetase. The results indicate that the cytoprotective effect of GSH on H 2O 2-induced cell injury in cultured bovine carotid artery endothelial cells depends on the increase in intracellular GSH content.