Synthesis Enzyme Activity Research Articles

Human umbilical cord-derived mesenchymal stem cells affect urea synthesis and the cell apoptosis of human induced hepatocytes by secreting IL-6 in a serum-free co-culture system.

Bioartificial livers (BALs) are emerging as a potential supportive therapy for liver diseases. However, the maintenance of hepatocyte function and viability in vitro is a major challenge. Mesenchymal stem cells (MSCs) have attracted extensive attention for providing trophic support to hepatocytes, but only few studies have explored the interaction between human MSCs and human hepatocytes, and very little is known about the underlying molecular mechanisms whereby MSCs affect hepatocyte function, especially in serum-free medium (SFM). This study aims to explore the effects of human umbilical cord-derived MSCs (hUMSCs) on human-induced hepatocytes (hiHeps) function and viability, and know about the underlying molecular mechanism of interaction in SFM. The liver-specific function of hiHeps was evaluated by analysis of albumin secretion, urea synthesis, and metabolic enzyme activity. hiHeps apoptosis was mainly characterized by live/dead staining assay, JC-1 mitochondrial membrane potential assay, reactive oxygen species (ROS) generation, and cell apoptosis detection. The expression of related genes and proteins were measured by qRT-PCR and western blotting. The results indicate that co-culture with hUMSCs improved hiHep urea synthesis and reduced cell apoptosis compared to monoculture in SFM, and this effect was found to be mediated by secreted interleukin-6 (IL-6). Further, studies revealed that IL-6 reduced hiHep apoptosis via the activation of the JAK-Stat3-Ref-1 and JAK-Stat3-Bcl-2/Bax-Caspase3 pathways by binding to the IL-6 receptor. IL-6 also enhanced hiHep urea synthesis through the JAK-Akt-P53-ARG1 pathway. Finally, hiHep-specific functions were further prolonged and increased when co-cultured with hUMSCs on 3D polyethylene terephthalate (PET) fibrous scaffolds. The SFM co-culture strategy showed major advantages in maintaining hiHep function and viability in vitro, which is of great significance for the clinical application of hiHeps in BALs.

The features of metabolism and structural organization of dental system under conditions of experimental insulin resistance

Peculiarities of calcium homeostasis, the activity of energy synthesis enzymes and structural organization of the dental system in rats under conditions of insulin resistance were analyzed. It was found that impaired glucose tolerance is manifested by a decreased mineralizing ability of hard tissues of alveolar processes and teeth, accompanied by a decrease in calcium content in erythrocyte mass and alveolar processes and an increase in acid phosphatase activity in blood serum. In rats with insulin resistance a decrease in the activity of lactate- and succinate dehydrogenase was detected. Degenerative changes were observed in the bone thickness of the dental area of insulin resistant rats, which were accompanied by the development of hypercellularity of the osteogenic layer of periosteum. Reactive changes in the tooth pulp were manifested by a decrease in the area of its loose connective tissue. Hyperplastic changes with the formation of unexpressed acanthotic bands developed in the oral mucosa of experimental animals, the thickness of the basal and granular layers increased against the background of a decrease in the prickle layer of epitheliocytes. In experimental group we observed a decrease in the nuclear cytoplasmic index and an increase in keratin formation in the epithelium. A narrowing of the lumen of capillaries and arterioles of the dental area, hyperplasia of endothelial cells and an increased accumulation of glycoproteins, especially in small arteries, were also detected in experimental group. Thus, altered carbohydrate metabolism leads to the metabolic changes of teeth supporting apparatus and oral mucosa, aggravating the course of insulin resistance with the development of dental system pathology.

The Changes of Prooxidant-Antioxidant and Energy Homeostasis in Rates with Impaired Glucose Tolerance

Topicality.Violation of glucose utilization leads to disruption of all types of metabolic processes. Particularly dangerous in the prognostic aspect is the combination of carbohydrate metabolism disorders and other endocrine pathologies, in particular hypothyroid dysfunction.
 The objective of the study was to examine prooxidant-antioxidant changes ofblood serum, teeth pulp and oral mucosa in rats with impaired glucose tolerance on the background of iodine deficiency.
 Materials and Methods. The studies were carried on 60 female rats, which were divided into two research groups - rats with impaired glucose tolerance and insulin resistance on the background of iodine deficiency. Lipid peroxidation processes were evaluated due to the content of diene conjugates and active products that react with thiobarbituric acid in blood serum, teeth pulp and oral mucosa. Antioxidant defence of blood serum was characterized by catalase, ceruloplasmin, superoxide dismutase, glutathione peroxidase, glutathione reductase activity and iron transferrin saturation. Energy metabolism was examined by the activity of succinate dehydrogenase, malate dehydrogenase and lactate dehydrogenase in blood serum.
 Results.Impaired glucose tolerance was foundto lead to the activation of lipoperoxidation, mainly due to the accumulation of final products of lipid peroxidation in all studied tissues against the background of redistribution of the activity of antiradical enzymes. Under these experimental conditions, different changes in the activity of energy synthesis enzymes were observed. The development of combined endocrine pathology has led to more pronounced changes in the prooxidant-antioxidant system and significant suppression of serum dehydrogenase activity.
 Conclusions.The development of insulin resistance on the background of iodine deficiency causes the activation of oxygen-dependent processes in periodontal tissues against the background of reduced antiradical defence and disruption of energy synthesis system.

Differences in morphological and physiological features of citrus seedlings are related to Mg transport from the parent to branch organs

BackgroundIn this study, we aimed to test the hypothesis that magnesium (Mg) remobilization in citrus plants is regulated by Mg supply and contributes to differences in the growth of the parent and branch organs. Citrus seedlings were grown in sand under Mg deficient (0 mmol Mg2+ L−1, -Mg) and Mg sufficient (2 mmol Mg2+ L−1, + Mg) conditions. The effects on biomass, Mg uptake and transport, gas exchange and chlorophyll fluorescence, as well as related morphological and physiological parameters were evaluated in different organs.ResultsMg deficiency significantly decreased plant biomass, with a decrease in total plant biomass of 39.6%, and a greater than twofold decrease in the branch organs compared with that of the parent organs. Reduced photosynthesis capacity was caused by a decreased in pigment levels and photosynthetic electron transport chain disruption, thus affecting non-structural carbohydrate accumulation and plant growth. However, the adaptive responses of branch leaves to Mg deficiency were greater than those in parent leaves. Mg deficiency inhibited plant Mg uptake but enhanced Mg remobilization from parent to branch organs, thus changing related growth variables and physiological parameters, including protein synthesis and antioxidant enzyme activity. Moreover, in the principal components analysis, these variations were highly clustered in both the upper and lower parent leaves, but highly separated in branch leaves under the different Mg conditions.ConclusionsMg deficiency inhibits the growth of the parent and branch organs of citrus plants, with high Mg mobility contributing to differences in physiological metabolism. These findings suggest that Mg management should be optimized for sustainable citrus production.

Trichoderma harzianum induces resistance to root-knot nematodes by increasing secondary metabolite synthesis and defense-related enzyme activity in Solanum lycopersicum L

Trichoderma spp. can improve plant defense against diverse plant pathogens, including fungi, bacteria, viruses, and nematodes. Nonetheless, the underlying mechanisms of Trichoderma-induced enhanced resistance, particularly against plant-parasitic root-knot nematodes (RKNs), remain poorly understood. Here, we show that T. harzianum (TH) could effectively suppress RKN (Meloidogyne incognita) infestation with an RKN reduction percentage of 61.88% in tomato plants (Solanum lycopersicum L.). While RKN infections increased the levels of reactive oxygen species (ROS; H2O2 and O2•‾) and lipid peroxidation in tomato roots, colonization with TH significantly reduced the levels of ROS, malondialdehyde, and electrolyte leakage, which was associated with increased accumulation of multiple secondary metabolites such as flavonoids, phenols, lignin, and cellulose on 75 days after inoculation with M. incognita. Consistent with the content of secondary metabolites, the activities of enzymes related to plant defense and the expression of associated genes (PAL, C4H, 4CL, CAD, LPO, CCOMT, Tpx1, and G6PDH) significantly increased in TH + RKN-inoculated roots compared with that in only RKN-treated roots. Moreover, TH inoculation prior to RKN infestation significantly increased the activity of pathogenesis-related (PR) proteins such as β-1,3-glucanase, chitinase, protease, and amylase by 86.31%, 36.25%, 39.78%, and 17.06%, respectively, compared with the RKN-infested roots. Furthermore, TH significantly stimulated the levels of salicylic acid and jasmonic acid. These results indicate a positive role of TH in inducing resistance against the RKNs by promoting secondary metabolism and the activity and transcripts of defense-related enzymes in tomato roots. Our study unveiled critical mechanisms of Trichoderma-induced improved resistance to RKNs, which can be useful for developing sustainable agricultural practices.

Impaired Expression of GABA Signaling Components in the Alzheimer's Disease Middle Temporal Gyrus.

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter, playing a central role in the regulation of cortical excitability and the maintenance of the excitatory/inhibitory (E/I) balance. Several lines of evidence point to a remodeling of the cerebral GABAergic system in Alzheimer’s disease (AD), with past studies demonstrating alterations in GABA receptor and transporter expression, GABA synthesizing enzyme activity and focal GABA concentrations in post-mortem tissue. AD is a chronic neurodegenerative disorder with a poorly understood etiology and the temporal cortex is one of the earliest regions in the brain to be affected by AD neurodegeneration. Utilizing NanoString nCounter analysis, we demonstrate here the transcriptional downregulation of several GABA signaling components in the post-mortem human middle temporal gyrus (MTG) in AD, including the GABAA receptor α1, α2, α3, α5, β1, β2, β3, δ, γ2, γ3, and θ subunits and the GABAB receptor 2 (GABABR2) subunit. In addition to this, we note the transcriptional upregulation of the betaine-GABA transporter (BGT1) and GABA transporter 2 (GAT2), and the downregulation of the 67 kDa isoform of glutamate decarboxylase (GAD67), the primary GABA synthesizing enzyme. The functional consequences of these changes require further investigation, but such alterations may underlie disruptions to the E/I balance that are believed to contribute to cognitive decline in AD.

A liver-on-a-chip for hepatoprotective activity assessment.

Hepatoprotectant is critical for the treatment of liver disease. This study first reported the application of a liver chip in the hepatoprotective effect assessment. We first established a biomimetic sinusoid-on-a-chip by laminating four types of hepatic cell lines (HepG2, HUVEC, LX-2, and U937 cells) in a single microchannel with the help of laminar flow in the microchannel and some micro-fences. This chip was straightforward to fabricate and operate and was able to be long-term cultured. It also demonstrated better hepatic activity (cell viability, albumin synthesis, urea secretion, and cytochrome P450 enzyme activities) over the traditional planar cell culture model. Then, we loaded three hepatoprotectants (tiopronin, bifendatatum, and glycyrrhizinate) into the chip followed by the addition of acetaminophen as a toxin. We successfully observed the hepatoprotective effect of these hepatoprotectants in the chip, and we also found that bifendatatum predominantly reduced alanine transaminase secretion, tiopronin predominantly reduced lactate dehydrogenase secretion, and glycyrrhizinate predominantly reduced aspartate transaminase secretion, which revealed the different mechanisms of these hepatoprotectants and provided a clue for following molecular biological study of the protecting mechanism.

Salvia hispanica L. (chia) seed promotes body fat depletion and modulates adipocyte lipid handling in sucrose-rich diet-fed rats

The aim of this study was to analyze the effects of Salvia hispanica L. (chia) seed upon metabolic pathways that play a key role in adipose tissue lipid handling which could be involved in visceral adiposity reduction developed in rats fed a sucrose-rich diet (SRD). Male Wistar rats were fed with a reference diet (RD) -6 months- or SRD-3 months. Then, the last group was randomly divided into two subgroups. One subgroup continued receiving the SRD up to 6 months and the other was fed with a SRD where whole chia seed was incorporated as the source of dietary fat for the next 3 months (SRD + CHIA). Results showed that chia seed in the SRD-fed rat reduced the abdominal and thoracic circumferences, carcass fat content, adipose tissue weights, and visceral adiposity index. This was accompanied by an improvement in insulin sensitivity and plasma lipid profile. In epididymal adipose tissue, the decreased fat cell triglyceride content was associated with a reduction in both, FAT/CD 36 plasma membrane levels and the fat synthesis enzyme activities. There were not changes in oxidative CPT enzyme activities. PKCβ and the precursor and mature forms of SREBP-1 protein levels were decreased, while pAMPK was increased. Our findings suggest that chia seed supplementation can modulate essential pathways of lipid metabolism in adipose tissue, contributing to reduced visceral fat accumulation in SRD-fed rats.

The Role of the Brain in the Regulation of Peripheral Organs–Noradrenaline Sources in Neonatal Rats: Noradrenaline Synthesis Enzyme Activity

This work is aimed at studying the mechanisms of reciprocal humoral regulation of noradrenaline-producing organs in rats in the perinatal period of development. The activity of noradrenaline synthesis enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase was measured in the brain and adrenal glands 48 and 72 h after the injection of immunotoxin (anti-dopamine-beta-hydroxylase-saporin) into the rat brain ventricles. It was shown that, 48 h after the immunotoxin injection into the brain, the activity of tyrosine hydroxylase in the brain decreased; however, 72 h after the injection it reached the control levels. This fact indicates that noradrenaline synthesis in the survived neurons increases. In the adrenal glands, 72 h after the immunotoxin injection into the brain, the activity of dopamine-beta-hydroxylase increased. This points to a compensatory increase in the rate of noradrenaline synthesis in the adrenal glands when the synthesis of noradrenaline in the brain is inhibited.

Accelerated grain-filling rate increases seed size and grain yield of recent naked oat cultivars under well-watered and water-deficit conditions

Increased seed size has greater contribution in yield improvement in recent naked oat (Avena sativa L. subsp. nudisativa) cultivars than cultivars derived from landraces. However, the underlying grain-filling mechanisms associated with seed size and grain yield in naked oat have received limited study. Two field experiments and a pot experiment compared grain-filling mechanisms, seed size and grain yield of ten naked oat cultivars, including old (landraces, released before 1950s) and new (released since 2008) cultivars. In both well-watered and water-deficit conditions, the new cultivars had higher grain yields, higher thousand kernel weights (TKW) and higher grain numbers per spike, but fewer fertile spikes per unit area or per plant, and no significant differences in grain number per unit area or per plant, than old cultivars. The findings in both the field and pot experiments demonstrated that increased grain-filling rate, rather than duration, enhanced seed size (TKW) and grain yield in the new cultivars. The increased grain-filling rate in a new cultivar was associated with higher rates of endosperm cell division and higher number of endosperm cells, compared to that in an old cultivar, but there were no significant differences in starch synthesis enzyme activity and ABA concentration between the old and new cultivars. As a result of current breeding and selection, the increased the grain yield of new cultivars of naked oat is primarily due to increased seed size (increased sink size) arising from a greater number of endosperm cells per grain, and not because of increased enzyme or phytohormonal activity. The larger seed size is important in the production of oats for food rather than for forage.

Regulation of Erythroid Heme Synthesis By the Mitochondrial Clpx Unfoldase

Differentiating erythroid cells synthesize large quantities of heme for hemoglobinization. While the transcriptional regulation and enzymatic mechanisms of the heme synthetic enzymes are well characterized, we lack mechanistic understanding of how their protein stability, cofactor incorporation and functional interactions with mitochondrial housekeeping proteins are regulated. These mechanisms can rapidly alter the rate of heme synthesis in response to external stimuli and metabolic requirements, and are critical for heme regulation within a tissue-specific and developmental context. CLPX, a mitochondrial protein unfoldase best understood for its function in a proteasome-like enzyme complex with the peptidase CLPP (the CLPXP ATP-dependent protease) plays a central role in regulation of mitochondrial protein turnover, is one such heme regulatory protein. CLPX activates yeast ALAS, which catalyzes the committed step of the heme synthesis pathway, by facilitating the incorporation of its cofactor, PLP, and is required for erythroid heme synthesis in zebrafish (Kardon et al. Cell 2015). Paradoxically, it regulates the turnover of ALAS1 and ALAS2 protein in vertebrate cell lines and appears to regulate the heme synthesis downstream of ALAS (Kubota et al. JBC 2016, Yien et al. PNAS 2017). However, it is not known if vertebrate ALAS was activated by ALAS, or if the requirement for CLPX in vertebrate heme synthesis was caused its regulation of ALAS activity (Figure A). To dissect the roles of CLPX in erythroid heme synthesis, we knocked out Clpxand Clpp in murine erythroleukemia (MEL) cells and assayed the activity, stability, and steady state levels of the heme synthesis enzymes, ALAS2 and FECH, which colocalize with ALAS in the mitochondrial matrix. Consistent with previous observations, Clpx -/- MEL cells had a heme defect, while Clpp -/-cells did not (Figure B). However, in contrast to previous observations in the yeast model, CLPX is not required for ALAS activation in erythroid cells, but plays a key role in regulating ALAS2 turnover in concert with the CLPP peptidase (Figure C). During erythroid differentiation, CLPP protein levels are decreased, stabilizing ALAS2 protein (Figure D). Although differentiating Clpx -/-and Clpp -/- MEL cells did not demonstrate any changes in ALAS2 turnover, likely because steady-state levels of CLPP protein were already decreased (Figure E), we observed an increase in steady-state ALAS2 protein levels and a dramatic increase in ALAS2 enzyme activity. In vitro mitochondrial iron transport/heme synthesis assays revealed a heme defect in Clpx -/-MEL cells, suggesting that CLPX plays a role in mitochondrial iron metabolism. Collectively, these data suggest a complex, differentiation-stage specific regulation of heme synthesis by the CLPXP proteolytic complex (Figure F). As Clpx -/- mouse embryos die by about E9.5 (mousephenotype.org), we dissected the in vivo role of Clpxin erythropoiesis by analyzing the phenotypes of clpxa and clpxb mutant zebrafish obtained from ZIRC. To accomplish this, we crossed clpxa and clpxb mutant zebrafish into Tg(lcr:GFP) zebrafish line in which erythroid cells are fluorescently labeled with GFP (Ganis et al Dev Biol 2012). We observed that clpxa mutant zebrafish had an early erythropoietic defect at 24 hpf that resolved at 48hpf; this developmental defect was not observed in clpxbmutant zebrafish. Benzidine staining of heme in mutant zebrafish revealed that while clpxa was dispensable for erythroid heme synthesis, clpxb was required for erythroid hemoglobinization (Figure G). Lastly, clpxbzebrafish mutants continued to be developmentally delayed and did not survive past 5 dpf. Collectively, our observations in cell lines and in the zebrafish model demonstrate that Clpx is essential for the maintenance of differentiated erythroid cells, as well as for the differentiation of the erythroid lineage. The control of heme synthesis and erythroid development by CLPX reveals how mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with key steps in heme synthesis. Further, our studies provide important genetic tools for dissecting these regulatory components in isolation as well as within the in vivocontext of erythropoiesis. Figure Disclosures No relevant conflicts of interest to declare.

Mulching with plastic film improved the root quality of summer-sown sweet potato (Ipomoea batatas (L). Lam.) in northern China

The root quality of sweet potato cultivated during the summer season is poor in northern China; thus, this study was conducted to determine whether root quality could be improved through mulching with plastic film (MPF). The effect of MPF on root starch and its composition, the activity of starch synthesis enzymes, and other quality-related parameters were investigated in two purple flesh sweet potato cultivars, Jishu 18 and Ayamurasaki (Aya). The results indicated that root dry matter, anthocyanin content, adenosine triphosphate (ATP), and starch content were higher in both cultivars under the MPF treatment than those under the control treatment. The root adenosine diphosphate glucose pyrophosphorylase/uridine diphosphate glucose pyrophosphorylase (ADPGPPase/UDPGPPase) activity and adenosine triphosphatease (ATPase) activity were increased using MPF. However, under the MPF treatment, the amylose content, soluble sugar content, and granule-bound synthase (GBSS) activity increased in Jishu 18 but decreased in Aya, and the amylopectin content, protein content, and soluble starch synthase (SSS) activity decreased in Jishu 18 but increased in Aya. Therefore, MPF seems benifit to improve the quality of sweet potato, but the effects of this treatment condition may be dependent on the cultivar.

Implication of salt stress induces changes in pigment production, antioxidant enzyme activity, and qRT-PCR expression of genes involved in the biosynthetic pathway of Bixa orellana L.

The effect of salt stress on pigment synthesis and antioxidant enzyme activity as well as in the genes involved in the biosynthetic pathway of bixin was studied. The 14-day germinated seedlings of Bixa orellana were induced into the various NaCl concentration (0, 25, 50, 75, 100mM). After 45days, leaves were taken for pigment analysis, antioxidant assays, and gene expression analysis to study the response of salt stress. The pigment content such as chlorophyll level was increased upon salt stress with a reduction in total carotenoid clearly indicating the adaptability of plants towards the stressed state. The level of β-carotene was increased in the highest concentration of salt stress treatment. The secondary metabolites such as bixin and abscisic acid (ABA) content were also high in elevated concentration of salt-treated seedling than control. The antioxidant enzyme activity was increased with the highest dose of salt stress suggesting the antioxidant enzymes to protect the plant from the deleterious effects. The mRNA transcript gene of the carotenoid biosynthetic pathway such as phytoene synthase (PSY), 1-deoxyxylulose-5-phosphate synthase (DXS), phytoene desaturase (PDS), beta-lycopene cyclase (LCY-β), epsilon lycopene cyclase (LCY-ε), carboxyl methyl transferase (CMT), aldehyde dehydrogenase (ADH), lycopene cleavage dioxygenase (LCD), and carotenoid cleavage dioxygenase (CCD) showed differential expression pattern under salt stress. In addendum, we studied the co-expression network analysis of gene to assess the co-related genes associated in the biosynthesis pathway of carotenoid. From the co-expression analysis result showed, the LCY, PDS, and PSY genes were closely correlated with other genes. These finding may provide insight to the plants to exist in the stress condition and to improve the industrially important pigment production.

Ameliorative effect of alendronate against intracerebroventricular streptozotocin induced alteration in neurobehavioral, neuroinflammation and biochemical parameters with emphasis on Aβ and BACE-1

Alzheimer’s disease (AD) is the most prevalent age related neurodegenerative disorder manifested by progressive cognitive decline and neuronal loss in the brain, yet precise etiopathology of majority of sporadic or late-onset AD cases is unknown. AD is associated with various pathological events such as Aβ deposition due to BACE-1 induced cleavage of APP, neuroinflammation, increased cholesterol synthesis, cholinergic deficit and oxidative stress. It was found that bone drug, alendronate (ALN) that cross blood brain barrier inhibits brain cholesterol synthesis and AChE enzyme activity. As cholesterol modifying agents have been supposed to alter AD like pathologies, the current study was designed to investigate the possible neuroprotective and therapeutic potential of ALN against ICV STZ induced experimental sporadic AD (SAD) in mice in a non-cholesterol dependent manner, using donepezil (5 mg/kg) as a reference standard. The preliminary study was done by molecular modelling to identify the binding affinity of ALN with BACE-1 in silico. The prevention of cognitive impairment in mice induced by ICV STZ (3 mg/kg) infused on first and third day, by ALN (1.76 mg/kg p.o.) administered for 15 consecutive days was assessed through Spontaneous Alternation Behavior (SAB) and Morris water maze (MWM) test. Additionally, the protective effect of ALN was also observed by the reversal of altered levels of Aβ1-42, BACE-,1 neuroinflammatory cytokines, AChE activity and oxidative stress markers (except TBARS) in ICV-STZ infused mice. However, the findings of the present study imply the therapeutic potential of ALN against SAD-like complications.

Overexpression of SbSKIP, A Pre-mRNA splicing factor from Sorghum bicolor, enhances root growth and drought tolerance in Petunia hybrida

Abstract Pre-mRNA splicing factors have been shown to have important roles in understanding the response of plants to various abiotic stresses. Different splicing factors might have different roles in plant stress responses. One splicer factor, skip (Sloan-Kettering retrovirus interacting protein), has been shown to play an important role in the tolerance of plants to various abiotic stresses although its mechanism of action is unknown. SbSKIP (Sorghum bicolor skip) encodes an RNA metabolism-related protein that could improve tobacco’s drought tolerance of tobacco. There is uncertainty how SbSKIP would act in other species and it effect on potential target genes. We found that overexpression of SbSKIP in Petunia hybrida could promote the growth of lateral roots and enhance drought tolerance. After withholding water for 14 d, SbSKIP plants grew better than WT (wild-type) plants; WT plants leaves were severely wilted and died, whereas transgenic plants were turgid after 17 d of drought treatment. Water stressed transgenic plants overexpressed SbSKIP, increased leaf proline synthesis and antioxidant defense enzyme activities, and decreased the accumulation of MDA. Furthermore, SbSKIP also regulated stress-induced gene expression under drought stress. These results suggest that overexpression of SbSKIP improves the tolerance of petunia to drought stress by enhancing antioxidant defense enzymes that decrease the content of cell membrane peroxidation, and by regulating the expression of stress-induced genes and the synthesis of proline.

Combining mutations at genes encoding key enzymes involved in starch synthesis affects the amylose content, carbohydrate allocation and hardness in the wheat grain.

SummaryModifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end products. The starch synthesized in the grain of conventional wheats (Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes (GBSSI, SSIIa and SBEIIa) has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker‐assisted breeding exercise, has been used to generate three mutant lines that produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and nonstarch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross‐talk between the starch and carbohydrate synthesis pathways.

High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes

BackgroundBiotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient.ResultsFor optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed−1 of wax esters, containing 27–34 mol% oleyl oleate.ConclusionsThe overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.

Functional hepatocyte clusters on bioactive blend silk matrices towards generating bioartificial liver constructs.

An end stage liver disease called cirrhosis perturbs the self-healing ability and physiological functions of liver. Due to the scarcity of healthy donors, a functional in vitro hepatic construct retaining the liver-specific functions is in great demand for its prospects in bioartificial liver (BAL) and cell-based tissue engineering. Physicochemical attributes of a matrix influence the behavior of cultured hepatocytes in terms of attachment, morphology and functionality. Mulberry and non-mulberry silk fibroin presents unique amino acid sequence with difference in hydrophobicity and crystallinity. Considering this, the present study focuses on the development of a suitable three-dimensional (3D) bioactive matrix incorporating both mulberry silk fibroin and cell adhesion motif (RGD) rich non-mulberry silk fibroin. Porous silk blend scaffolds facilitated the formation of hepatocyte clusters with enhanced liver-specific functions emphasizing both cell-cell and cell-matrix interactions. Hemocompatibility and integral property of blend scaffolds offers a biological niche for seeding functional liver cells that would have future prospects in biohybrid devices.

A perfusion incubator liver chip for 3D cell culture with application on chronic hepatotoxicity testing

Liver chips have been developed to recapitulate in vivo physiological conditions to enhance hepatocyte functions for assessing acute responses to drugs. To develop liver chips that can assess repeated dosing chronic hepatotoxicity, we need to ensure that hepatocyte functions be maintained at constant values over two weeks in stable culture conditions of sterility, temperature, pH, fluidic-flow of culture media and drugs. We have designed a perfusion-incubator-liver-chip (PIC) for 3D cell culture, that assures a tangential flow of the media over the spheroids culture. Rat hepatocyte spheroids constrained between a cover glass and a porous-ultrathin Parylene C membrane experienced optimal mass transfer and limited shear stress from the flowing culture media; maintained cell viability over 24 days. Hepatocyte functions were significantly improved and maintained at constant values (urea, albumin synthesis, and CYP450 enzyme activities) for 14 days. The chip act as an incubator, having 5% CO2 pressure-driven culture-media flow, on-chip heater and active debubbler. It operates in a biosafety cabinet, thus minimizing risk of contamination. The chronic drug response to repeated dosing of Diclofenac and Acetaminophen evaluated in PIC were more sensitive than the static culture control.

The Proper Supply of S Increases Amino Acid Synthesis and Antioxidant Enzyme Activity in Tanzania Guinea Grass Used for Cd Phytoextraction

Increased Cd concentrations in the environment impair plant growth, but plants properly supplied with S may develop greater tolerance to the damage caused by Cd and be used in the remediation of contaminated environments. The aim of this study was to evaluate the Cd-phytoextraction potential of Panicum maximum cv. Tanzania grown with S rates and to identify alterations in the concentrations of nutrients and amino acids and in the activity of some antioxidant enzymes under Cd stress conditions. Combinations of five S rates (0.1, 1.0, 1.9, 2.8, and 3.7 mmol L−1) and five Cd rates (0.0, 0.5, 1.0, 1.5, and 2.0 mmol L−1) in a nutrient solution were provided in two plant growth periods. Concentrations of N, P, and Zn increased, while K, Fe, and Mn decreased with exposure to Cd. The concentration of Ca decreased as the S supply was increased. Isoleucine, leucine, proline, and valine concentrations increased with exposure to Cd and with higher levels of S. The APX activity was higher at the highest Cd exposure level. Activity and number of SOD and GR isoforms in the roots and of CAT in the shoots of the regrown plant decreased at the highest level of contamination by Cd, which was lessened by the supply of greater S rates. Tanzania guinea grass grown with an adequate supply of S has the potential for phytoextraction of Cd-contaminated environments.