Enzyme mRNA Research Articles

Artemisinin derivative DHA27 enhances the antibacterial effect of aminoglycosides against Pseudomonas aeruginosa by inhibiting mRNA expression of aminoglycoside-modifying enzymes.

Bacterial resistance is becoming increasingly serious, the present study aimed to investigate the mechanism of antibacterial sensitization effect of DHA27 combined with tobramycin in tobramycin-resistant Pseudomonas aeruginosa (PA). We found that DHA27 combined with aminoglycosides had an antibacterial sensitization effect on PA. Tobramycin, owing to its lower toxic and side effects, was selected to further study the molecular mechanism of drug combination. A sublethal-dose bacterial challenge/sepsis mouse model was established to study the protective effect of DHA27 plus tobramycin. Scanning electron microscopy was used to investigate whether DHA27 exerts the antibacterial sensitization effect by directly affecting bacterial morphology. The effect of DHA27 on daunorubicin accumulation in bacteria was studied, and quantitative reverse transcription PCR was used to study the effect of DHA27 plus tobramycin on 16S rRNA methyltransferase and aminoglycoside-modifying enzyme mRNA expression. Twenty clinical isolates of PA were found to be tobramycin resistant; DHA27 plus tobramycin had a significant antibacterial sensitization effect on many of these resistant strains. DHA27 plus tobramycin reduced the bacterial load in the spleen and lungs of sepsis model mice and levels of proinflammatory cytokines interleukin-1β (IL-1β) and interferon-γ (IFN-γ). DHA27 plus tobramycin significantly inhibited the mRNA expression of aminoglycoside-modifying enzymes in bacteria. DHA27 combined with AGs had an antibacterial sensitization effect on PA; the molecular mechanism underlying this effect is closely related to the inhibition of the mRNA expression of aminoglycoside-modifying enzymes, especially aac(3)-II.

Expression and localization of NRF2/Keap1 signalling pathway genes in mouse preimplantation embryos exposed to free fatty acids

Obese women experience greater incidence of infertility, with reproductive tracts exposing preimplantation embryos to elevated free fatty acids (FFA) such as palmitic acid (PA) and oleic acid (OA). PA treatment impairs mouse preimplantation development in vitro, while OA co-treatment rescues blastocyst development of PA treated embryos. In the present study, we investigated the effects of PA and OA treatment on NRF2/Keap1 localization, and relative antioxidant enzyme (Glutathione peroxidase; Gpx1, Catalase; Cat, Superoxide dismutase; Sod1 and γ-Glutamylcysteine ligase catalytic unit; Gclc) mRNA levels, during in vitro mouse preimplantation embryo development. Female mice were superovulated, mated, and embryos cultured in the presence of bovine Serum albumin (BSA) control or PA, or OA, alone (each at 100 μM) or PA + OA combined (each at 100 μM) treatment. NRF2 displayed nuclear localization at all developmental stages, whereas Keap1 primarily displayed cytoplasmic localization throughout control mouse preimplantation development in vitro. Relative transcript levels of Nrf2, Keap1, and downstream antioxidants significantly increased throughout control mouse preimplantation development in vitro. PA treatment significantly decreased blastocyst development and the levels of nuclear NRF2, while OA and PA + OA treatments did not. PA and OA treatments did not impact relative mRNA levels of Nrf2, Keap1, Gpx1, Cat, Sod1 or Gclc. Our outcomes demonstrate that cultured mouse embryos display nuclear NRF2, but that PA treatment reduces nuclear NRF2 and thus likely impacts NRF2/KEAP1 stress response mechanisms. Further studies should investigate whether free fatty acid effects on NRF2/KEAP1 contribute to the reduced fertility displayed by obese patients.

The development, reproduction and P450 enzyme of the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae) under the sublethal concentrations of clothianidin

Clothianidin is a second-generation neonicotinoid insecticide that can effectively prevent piercing-sucking pests, such as white-backed planthopper (WBPH), Sogatella furcifera (Horváth). In this study, the sublethal effects of clothianidin on the biological traits of S. furcifera were evaluated via the age-stage, two-sex life table procedure. Our results exhibited that the female adult longevity, fecundity and hatchability of F0 generation were significantly decreased after LC10 and (or) LC30 of clothianidin exposure compared to the control. Transgenerational effects showed that the pre-adult period, female adult longevity, total longevity, oviposition days (Od), fecundity and mean generation time (T) of F1 generation were significantly decreased in the LC10 and LC30 groups compared to the control. Moreover, the development times of the third- and fifth-instar nymphs, total preoviposition period (TPOP) and doubling time (DT) were significantly shorter in the LC10 group than in the control and LC30 groups. Furthermore, the intrinsic rate of increase (ri) and finite rate of increase (λ) values of the LC10 group were significantly higher than those of the control group. However, there was no significant difference in the male adult longevity, adult preoviposition period (APOP) and net reproductive rate (R0) between the treated groups and the control. Enzyme activity and gene expression results showed that the P450 enzyme activity and mRNA levels of many P450 genes were significantly increased by clothianidin treatment. In addition, the knockdown of CYP4CE3 and CYP6FJ3, which showed the highest inducing levels, by RNA interference (RNAi) dramatically increased the toxicity of clothianidin against S. furcifera. These results indicated that sublethal concentrations of clothianidin showed a stimulatory effect on the development, but it could adversely affect the survival and reproduction of S. furcifera. Additionally, CYP4CE3 and CYP6FJ3 might play an important role in the detoxification and evolution of clothianidin resistance in S. furcifera.

Effects of Aloe vera extract on growth, viability, ultrastructure and expression of mRNA for antioxidant enzymes in bovine secondary follicles cultured in vitro

This study aimed to investigate the effects of Aloe vera extract on follicular growth, viability, ultrastructure, and mRNA levels for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, secondary follicles were mechanically isolated from the ovarian cortex and cultured at 38.5 °C, with 5% CO2 in air, for 18 days in TCM-199+ alone or supplemented with 2.5%, 5.0%, 10.0% and 20.0% Aloe vera extract. Follicular growth, morphology and antrum formation were evaluated every 6 days, while ultrastructure was evaluated at the end of culture. Analysis of viability was performed by calcein-AM and ethidium homodimer-1, while mRNA levels for SOD, CAT, GPX1 and PRDX6 were evaluated by real-time PCR at the end of culture. The results show that follicles cultured with 2.5% Aloe vera had increased the rate of antrum formation, while 2.5% and 5.0% Aloe vera improved follicular viability rate. Follicles cultured with 2.5% and 10.0% Aloe vera increased the levels of mRNA for SOD and GPX1 respectively, but the levels of CAT were reduced in follicles cultured with 2.5%, 5.0%, 10.0% and 20.0%. Additionally, follicles cultured with 2.5% of Aloe vera had their ultrastructure well preserved, while those cultured with 5.0%, 10.0% and 20.0% exhibited increased oocyte vacuolization and damaged organelles. In conclusion, 2.5% Aloe vera increases antrum formation, viability and expression of mRNA for SOD in cultured secondary follicles, but higher concentrations of Aloe vera have negative effects on follicular ultrastructure.

Heat-Killed Lacticaseibacillus paracasei Ameliorated UVB-Induced Oxidative Damage and Photoaging and Its Underlying Mechanisms.

Ultraviolet B (UVB) radiation is a major environmental causative factor of skin oxidative damage and photoaging. Lacticaseibacillus paracasei is a well-known probiotic strain that can regulate skin health. The present study investigated the effects of heat-killed Lacticaseibacillus paracasei (PL) on UVB linked oxidative damage and photoaging in skin cells (Normal human dermal fibroblast (NHDF) cells and B16F10 murine melanoma cells). Results demonstrated that: (1) PL prevented UVB-induced cytotoxicity relating to decreased DNA damage in NHDF and B16F10 cells; (2) PL alleviated UVB-induced oxidative damage through increasing GSH content, as well as antioxidant enzyme activities and mRNA levels (except MnSOD activity and mRNA levels as well as CAT mRNA level) relating to the activation of Sirt1/PGC-1α/Nrf2 signaling in NHDF cells; (3) PL attenuated UVB-induced photoaging was noticed with a decrease in the percentage of SA-β-gal positive cells in NHDF cells model. Moreover, PL attenuated UVB-induced photoaging through exerting an anti-wrinkling effect by enhancing the type I collagen level relating to the inhibition (JNK, p38)/(c-Fos, c-Jun) of signaling in NHDF cells, and exerting an anti-melanogenic effect by suppressing tyrosinase and TYRP-1 activity and/or expressions relating to the inhibition of PKA/CREB/MITF signaling in B16F10 cells. In conclusion, PL could ameliorate UVB-induced oxidative damage and photoaging. Therefore, PL may be a potential antioxidant and anti-photoaging active ingredient for the cosmetic industry.

Sexual Dimorphism in the Expression of Cytochrome P450 Enzymes in Rat Heart, Liver, Kidney, Lung, Brain, and Small Intestine.

Cytochrome P450 (P450) enzymes are monooxygenases that are expressed hepatically and extrahepatically and play an essential role in xenobiotic metabolism. Substantial scientific evidence indicates sex-specific differences between males and females in disease patterns and drug responses, which could be attributed, even partly, to differences in the expression and/or activity levels of P450 enzymes in different organs. In this study, we compared the mRNA and protein expression of P450 enzymes in different organs of male and female Sprague-Dawley rats by real-time polymerase chain reaction and western blot techniques. We found significant sex- and organ-specific differences in several enzymes. Hepatic Cyp2c11, Cyp2c13, and Cyp4a2 showed male-specific expression, whereas Cyp2c12 showed female-specific expression. Cyp2e1 and Cyp4f enzymes demonstrated higher expression in the female heart and kidneys compared with males; however, they showed no significant sexual dimorphism in the liver. Male rats showed higher hepatic and renal Cyp1b1 levels. All assessed enzymes were found in the liver, but some were not expressed in other organs. At the protein expression level, CYP1A2, CYP3A, and CYP4A1 demonstrated higher expression levels in the females in several organs, including the liver. Elucidating sex-specific differences in P450 enzyme levels could help better understand differences in disease pathogeneses and drug responses between males and females and thus improve treatment strategies. SIGNIFICANCE STATEMENT: This study characterized the differences in the mRNA and protein expression levels of different cytochrome P450 (P450) enzymes between male and female rats in the heart, liver, lung, kidney, brain, and small intestine. It demonstrated unique sex-specific differences in the different organs. This study is considered a big step towards elucidating sex-specific differences in P450 enzyme levels, which is largely important for achieving a better understanding of the differences between males and females in the disease's processes and treatment outcomes.

Ameliorative effect of bayberry leaves proanthocyanidins on high sugar diet induced Drosophila melanogaster.

Bayberry leaves proanthocyanidins (BLPs) were distributed in natural plant food, considered to have the potential for metabolic syndrome. In this study, we raised Drosophila melanogaster on high sugar diet (HSD) from the egg stage to induce hyperglycemia, and the ameliorative effect of BLPs was assessed based on this model. Phenotypical, biochemical, and molecular analyses related to diabetes mellitus pathogenesis were measured. Flies exposed to BLPs were found to suppress the HSD-induced high glucose and high triglycerides levels. Moreover, BLPs showed an inhibitory effect on carbohydrate digestive enzymes (α-amylase and α-glucosidase) activity and mRNA expression, exhibiting the potential for carbohydrate digestion retardation. Transcriptional levels of key genes associated with glycolipid metabolism were further evaluated, including dilp, InR, and downstream dAKT-dFOXO-PEPCK, together with E78, SREBP, FAS, and LSD genes, were all downregulated after BLPs-exposure, suggesting the ameliorative effect of BLPs on dysbiosis associated with the insulin signaling pathway. This study provided a new functional compound, which is beneficial to further antidiabetic therapy studies.

Ergosterol increases 7-dehydrocholesterol, a cholesterol precursor, and decreases cholesterol in human HepG2 cells.

Current treatment approaches for hyperlipidemia rely mainly on reducing the cholesterol level by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which is involved in the presqualene pathway of cholesterol biosynthesis. Finding a compound that instead targets the postsqualene pathway could aid in the treatment of hyperlipidemia and synergistically reduce the cholesterol level when used in conjunction with HMGCR inhibitors. Ergosterol is a fungal sterol that is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7). DHCR7 is also a cholesterol biosynthesis enzyme, and thus ergosterol may cause the accumulation of 7-dehydrocholesterol, a precursor of cholesterol and vitamin D3 , by a competitive effect. In this study, we examined the effect of ergosterol on the postsqualene pathway by quantifying cholesterol precursors and related sterols using gas chromatography-mass spectrometry and by conducting quantitative RT-PCR and western blot analysis for human HepG2 hepatoma cells. We found that ergosterol is converted into brassicasterol by the action of DHCR7 from HepG2 cells and that it induced the accumulation of cholesterol precursors (lathosterol, 7-dehydrocholesterol, and desmosterol) and decreased the cholesterol level by altering the mRNA and protein levels of cholesterol biosynthesis enzymes (increase of sterol 8,7-isomerase [EBP] and decrease of DHCR7 and 24-dehydrocholesterol reductase [DHCR24]). These results demonstrate that ergosterol inhibits the postsqualene pathway and may be useful for the prevention of hyperlipidemia.

Attainment of Sexual Maturity and Gonadotropin Priming in Gilts Determine Follicular Development, Endocrine Milieu and Response to Ovulatory Triggers.

The routine procedure of estrous cycle synchronization in pigs allows for the use of gonadotropins to stimulate ovarian activity. The applied protocols of eCG and hFSH priming similarly affected development of ovarian follicles in two classes 3–6 mm and >6 mm of diameter, however, the number of small follicles (<3 mm) was 2-fold higher in hFSH- than in eCG-primed prepubertal gilts. The attainment of sexual maturity increased concentration of estradiol, testosterone and androstenedione in the follicular fluid of hFSH/eCG-primed gilts, however, prostaglandin E2 and F2α metabolite increased in mature hFSH- and eCG-primed gilts, respectively. The maturity increased mRNA and/or protein expression of key steroidogenic enzymes, prostaglandin synthases or luteinizing hormone receptors in follicular walls. Both hormonal primers played a moderate role in affecting expression of steroidogenic enzymes in follicular walls. In vitro studies showed higher estradiol production in r-hLH (p = 0.04)- and r-hCG (p = 0.049)-stimulated follicular walls of mature gilts than in prepubertal hFSH-primed gilts. Both ovulatory triggers decreased the abundance of LHCG/FSH mRNA receptors in follicular walls, which mimic downregulation of these receptors by a preovulatory LH surge, confirmed in vivo. These data revealed the importance of sexual maturity in the protection of the estrogenic environment, and the selective, moderate role of eCG and FSH in the activation of steroidogenic enzymes in preovulatory follicles.

Arginine Enhances Ovarian Antioxidant Capability via Nrf2/Keap1 Pathway during the Luteal Phase in Ewes.

Simple SummaryThe quantity and quality of forage grass may decrease to some extent in autumn and winter, and feeding with such forage grass may temporarily cause malnutrition and ovarian oxidative stress in ewes. Arginine is one of the most abundant amino acids enhancing the antioxidant capability in animal tissues. Thus, this study investigated the effects of arginine on ovarian antioxidant capability during the luteal phase in ewes. Our results showed that nutrition restriction can reduce the ovarian antioxidant capability in ewes, while arginine supplementation can significantly improve it, which was associated with the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 pathway.This study evaluated the effect of arginine (Arg) on ovarian antioxidant capability during the luteal phase in ewes. A total of 108 multiparous Hu sheep at two years of age were randomly allocated to three groups: a control group (CG), a restriction group (RG), and an Arg group (AG), with six replicates per group and six ewes per replicate. Our results showed that the end body weight was significantly decreased in the RG group (p < 0.05), while the Arg addition reversed this reduction. The estrous cycle days were significantly increased in the RG group (p < 0.05), while Arg addition reversed this time extension. Compared with the control group, restricting feeding could significantly enhance the number of small follicles (SF), total follicles (TF), large corpora lutea, and the SF/TF (p < 0.05), while Arg addition reduced the number of SF and TF. However, the large follicles/TF were significantly decreased (p < 0.05), while Arg addition reversed this reduction. In addition, nutrition restriction significantly increased the malondialdehyde (MDA) level (p < 0.05), while significantly decreased the glutathione/glutathione disulfide and the activities of superoxidative dismutase, catalase, and glutathione peroxidase in the ovaries (p < 0.05). However, Arg addition reversed this enhancement of the MDA level and the reductions in these antioxidant enzymes activities. In addition, positive relationships occurred between antioxidant enzyme activities and the enzyme mRNA expressions. Meanwhile, the nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression was positively connected with antioxidant mRNA expressions and negatively related to the Kelch-like ECH-associated protein 1 (Keap1) mRNA expression. The Nrf2 protein expression was negatively related to the Keap1 protein expression. In conclusion, nutrition restriction reduced the ovarian antioxidant capability in ewes, while this was significantly improved by Arg supplementation, which was associated with the Nrf2/Keap1 pathway.

LAMP-1 Chimeric to HIV-1 p55Gag in the Immunization of Neonate Mice Induces an Early Germinal Center Formation and AID Expression.

Neonates have a limited adaptive response of plasma cells, germinal center (GC) B cells, and T follicular helper cells (TFH). As neonatal vaccination can be an important tool for AIDS prevention, these limitations need to be overcome. Chimeric DNA vaccine encoding p55Gag HIV-1 protein conjugated with lysosomal-associated membrane protein 1 (LAMP-1) has been described as immunogenic in the neonate period. Herein, we investigated the immunologic mechanisms involved in neonatal immunization with a LAMP-1/p55Gag (LAMP/Gag) DNA vaccine in a C57BL/6 mouse background. Neonatal LAMP/Gag vaccination induced strong Gag-specific T-cell response until adulthood and elevated levels of anti-Gag IgG antibodies. We also demonstrated for the first time that the immunogenicity of the neonatal period with LAMP/Gag is due to the induction of high-affinity anti-p24 IgG antibodies and long-term plasma cells. Together with that, there is the generation of early TFH cells and the formation of GC sites with the upregulation of activation-induced cytidine deaminase (AID) enzyme mRNA and protein expression in draining lymph nodes after neonatal LAMP/Gag vaccination. These findings underscore that the LAMP-1 strategy in the chimeric vaccine could be useful to enhance antibody production even in the face of neonatal immaturity, and they contribute to the development of new vaccine approaches for other emerging pathogens at an early stage of life.

SRNA-controlled iron sparing response in Staphylococci.

Staphylococcus aureus, a human opportunist pathogen, adjusts its metabolism to cope with iron deprivation within the host. We investigated the potential role of small non-coding RNAs (sRNAs) in dictating this process. A single sRNA, named here IsrR, emerged from a competition assay with tagged-mutant libraries as being required during iron starvation. IsrR is iron-repressed and predicted to target mRNAs expressing iron-containing enzymes. Among them, we demonstrated that IsrR down-regulates the translation of mRNAs of enzymes that catalyze anaerobic nitrate respiration. The IsrR sequence reveals three single-stranded C-rich regions (CRRs). Mutational and structural analysis indicated a differential contribution of these CRRs according to targets. We also report that IsrR is required for full lethality of S. aureus in a mouse septicemia model, underscoring its role as a major contributor to the iron-sparing response for bacterial survival during infection. IsrR is conserved among staphylococci, but it is not ortholog to the proteobacterial sRNA RyhB, nor to other characterized sRNAs down-regulating mRNAs of iron-containing enzymes. Remarkably, these distinct sRNAs regulate common targets, illustrating that RNA-based regulation provides optimal evolutionary solutions to improve bacterial fitness when iron is scarce.

Sulforaphane Increase Mitochondrial Biogenesis-Related Gene Expression in the Hippocampus and Suppresses Age-Related Cognitive Decline in Mice.

Sulforaphane (SFN) is a potent activator of the transcriptional factor, Nuclear Factor Erythroid 2 (NF-E2)-Related factor 2 (NRF2). SFN and its precursor, glucoraphanin (sulforaphane glucosinolate, SGS), have been shown to ameliorate cognitive function in clinical trials and in vivo studies. However, the effects of SGS on age-related cognitive decline in Senescence-Accelerated Mouse Prone 8 (SAMP8) is unknown. In this study, we determined the preventive potential of SGS on age-related cognitive decline. One-month old SAMP8 mice or control SAM resistance 1 (SAMR1) mice were fed an ad libitum diet with or without SGS-containing broccoli sprout powder (0.3% w/w SGS in diet) until 13 months of age. SGS significantly improved long-term memory in SAMP8 at 12 months of age. Interestingly, SGS increased hippocampal mRNA and protein levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α) and mitochondrial transcription factor A (TFAM), which are master regulators of mitochondrial biogenesis, both in SAMR1 and SAMP8 at 13 months of age. Furthermore, mRNAs for nuclear respiratory factor-1 (NRF-1) and mitochondrial DNA-encoded respiratory complex enzymes, but not mitochondrial DNA itself, were increased by SGS in SAMP8 mice. These results suggest that SGS prevents age-related cognitive decline by maintaining mitochondrial function in senescence-accelerated mice.

Effects of Dietary Amylose-Amylopectin Ratio on Growth Performance and Intestinal Digestive and Absorptive Function in Weaned Piglet Response to Lipopolysaccharide.

Simple SummaryAs one of the most important components, starch has made great contributions to feed but was wasted numerously during pig feeding. This paper is designed to explore the optimal ratio of dietary amylose to amylopectin for gut health and absorption, thereby improving starch utilization. Our results indicated that the dietary amylose/amylopectin ratio (AAR) of 0.60 could reduce feed conversion rate of piglets, having a certain positive significance for saving feed. In addition, under lipopolysaccharide (LPS) stress, a diet with an AAR of 0.40 to 0.60 significantly improved the intestinal health of piglets, which would provide data to support for the formulation of feed in weaned piglets during bacterial infection.This study investigated the effects of diet with different amylose–amylopectin ratios (AAR) on the growth performance, intestinal morphology, digestive enzyme activities and mRNA expression of nutrients transporters in piglets with short-term lipopolysaccharide (LPS) intraperitoneal injections. Sixty 21 days-old piglets (Landrace × Yorkshire; 6.504 ± 0.079) were randomly assigned based on their body weight (BW) and litters of origins to five groups with experimental diets with an AAR of 0.00, 0.20, 0.40, 0.60, or 0.80 (namely, the 0.00, 0.20, 0.40, and 0.80 groups), respectively. Each treatment included 12 piglets (one piglet per pen). This experiment lasted for 28 days. On the 28th day, six piglets in each treatment were randomly selected for an LPS intraperitoneal injection (100 μg/kg BW), and other piglets were injected with normal saline. Twelve hours after LPS injection, all piglets were sacrificed to collect small intestinal mucosa for analysis. Although different AAR did not influence the final BW in piglets, the piglets in the 0.40 group represented the poorest feed-to-gain ratio (F/G) in the first, second and fourth week (p < 0.05) and the lowest average daily gain (ADG) in the fourth week (p < 0.05) compared with other groups. In terms of the small intestinal morphology, piglets in the 0.20 and 0.60 groups showed better ileal villous width (p < 0.05). Piglets in the 0.60 group presented greater activities of jejunal maltase, sucrase and alkaline phosphatase (p < 0.05) than those of 0.20 and 0.40. However, a low amylose diet increased the mRNA expression of jejunal glucose and amino acid transporters (p < 0.05). In addition, compared to saline injection, the LPS challenge significantly lessened jejunal digestive enzyme activities (p < 0.01) and, ileal villous width and downregulated the gene expression of glucose and amino acid transporters (p < 0.05) in piglets. Interestingly, certain diet -LPS interactions on duodenal VH/CD, jejunal maltase activity (p < 0.05) and the expression of glucose transporters (p < 0.05) were observed. Taken together, in terms of small intestinal digestion and absorption capacity, these results demonstrated that a diet with an AAR of 0.60 diets could improve the intestinal digestive and absorptive capability by affecting small intestinal morphology, digestive enzymes, and nutrients absorptions in piglets. In addition, the diets containing an AAR of 0.40–0.60 were more likely to resist the damage of LPS stress to intestinal morphology and nutrient absorption.

Sex-Dependent Alterations in the mRNA Expression of Enzymes Involved in Dopamine Synthesis and Breakdown After Methamphetamine Self-Administration.

Sex differences have been reported in methamphetamine (METH) use disorder in humans and in animal models of METH exposure. Specifically, animals that self-administer METH show sex-related dissimilarities in dopamine (DA) metabolism. To better understand the molecular bases for the differences in DA metabolism, we measured the levels of mRNAs of enzymes that catalyze DA synthesis and breakdown in the prefrontal cortex (PFC), nucleus accumbens (NAc), dorsal striatum (dSTR), and hippocampus (HIP) of rats that had self-administered METH. There were significant sex differences in control rats, with males having higher basal levels of Th in the PFC and dSTR, Ddc in the NAc, and MaoB in the HIP. In contrast, female controls showed higher basal levels of Comt in the HIP. Male and female METH SA rats also showed some distinct responses to the drug. Specifically, female METH rats exhibited increased expression of Ddc and MaoB, whereas male METH animals showed higher levels of Comt mRNA in the PFC compared to their respective controls. In the NAc, male METH rats displayed decreased Th and Ddc mRNA levels. Together, our results identified sex-dependent and region-specific changes in the mRNA expression of several enzymes involved in DA synthesis and breakdown in response to METH SA, with the majority of differences being observed in the mesocorticolimbic dopaminergic system. These findings are of significant translational importance providing further support for the inclusion of sex as an important variable when planning and evaluating therapeutic interventions against METH use disorder in human clinical studies.

Lipid metabolism and m6A RNA methylation are altered in lambs supplemented rumen-protected methionine and lysine in a low-protein diet

BackgroundMethionine or lysine has been reported to influence DNA methylation and fat metabolism, but their combined effects in N6-methyl-adenosine (m6A) RNA methylation remain unclarified. The combined effects of rumen-protected methionine and lysine (RML) in a low-protein (LP) diet on lipid metabolism, m6A RNA methylation, and fatty acid (FA) profiles in the liver and muscle of lambs were investigated. Sixty-three male lambs were divided into three treatment groups, three pens per group and seven lambs per pen. The lambs were fed a 14.5% crude protein (CP) diet (adequate protein [NP]), 12.5% CP diet (LP), and a LP diet plus RML (LP + RML) for 60 d.ResultsThe results showed that the addition of RML in a LP diet tended to lower the concentrations of plasma leptin (P = 0.07), triglyceride (P = 0.05), and non-esterified FA (P = 0.08). Feeding a LP diet increased the enzyme activity or mRNA expression of lipogenic enzymes and decreased lipolytic enzymes compared with the NP diet. This effect was reversed by supplementation of RML with a LP diet. The inclusion of RML in a LP diet affected the polyunsaturated fatty acids (PUFA), n-3 PUFA, and n-6 PUFA in the liver but not in the muscle, which might be linked with altered expression of FA desaturase-1 (FADS1) and acetyl-CoA carboxylase (ACC). A LP diet supplemented with RML increased (P < 0.05) total m6A levels in the liver and muscle and were accompanied by decreased expression of fat mass and obesity-associated protein (FTO) and alkB homologue 5 (ALKBH5). The mRNA expressions of methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) in the LP + RML diet group were lower than those in the other two groups. Supplementation of RML with a LP diet affected only liver YTH domain family (YTHDF2) proteins (P < 0.05) and muscle YTHDF3 (P = 0.09), which can be explained by limited m6A-binding proteins that were mediated in mRNA fate.ConclusionsOur findings showed that the inclusion of RML in a LP diet could alter fat deposition through modulations of lipogenesis and lipolysis in the liver and muscle. These changes in fat metabolism may be associated with the modification of m6A RNA methylation.Graphical abstractA systematic graph illustrates the mechanism of dietary methionine and lysine influence on lipid metabolism and M6A. The green arrow with triangular heads indicates as activation and brown-wine arrows with flat heads indicates as suppression.

Inhibition of imrecoxib on mRNA and protein expression of CYP2C11 enzyme in rats.

To evaluate the effect of imrecoxib on CYP2C11 enzyme activity, mRNA, and protein expression, a UPLC method was established. Tolbutamide was selected as the CYP2C11 enzyme-specific probe drug and incubated with imrecoxib in rat liver microsomes. The yield of 4-hydroxytolbutamide was measured using UPLC to investigate the effect of imrecoxib on CYP2C11 enzyme activity. Imrecoxib (10mg/kg) was administered intragastrically twice daily. After 1, 7, and 14 days of administration, the liver tissues were analyzed. The expression of CYP2C11 enzyme mRNA was determined using reverse transcription-polymerase chain reaction, and its protein expression was determined using Western blot analysis. Imrecoxib concentration was inversely proportional to the production of 4-hydroxytolbutamide in liver microsomes. Imrecoxib demonstrated a dose-dependent inhibitory effect on CYP2C11 activity with IC50 = 74.77 μM. After administration, reverse transcription-polymerase chain reaction showed CYP2C11 enzyme mRNA expressions were 65% (P < 0.05), 35%, and 34% of the control group, respectively (P < 0.01). Western blot analysis showed CYP2C11 enzyme protein expressions were 80, 37, and 34% of the control group, respectively (P < 0.01). Imrecoxib can reduce mRNA and protein expression of CYP2C11 enzyme in rat liver and inhibit the activity of CYP2C11 enzyme in a dose-dependent manner. However, it does not produce clinically significant drug interactions.

Therapy Follows Diagnosis: Old and New Approaches for the Treatment of Acute Porphyrias, What We Know and What We Should Know.

Heme, iron protoporphyrin IX, is one of life’s most central molecules. Hence, availability of the enzymatic machinery necessary for its synthesis is crucial for every cell. Consequently, inborn errors of porphyrin metabolism that compromise normal synthesis, namely the family of porphyrias, undermine normal cellular metabolism given that heme has functions in catalytic centers, signal transduction and functional regulation and its synthesis is fully integrated into the center of intermediary metabolism. Very often, diagnosis of porphyrias is difficult and therefore delayed. Therapy can be as complicated. Over the last 50 years, several strategies have been developed: because of its integration with other parts of intermediary metabolism, the infusion of glucose (glucose effect) was one of the first attempts to counterbalance the dysregulation of porphyrin synthesis in porphyrias. Since heme synthesis is impaired, infusional replacement of heme was the next important therapeutic step. Recently, siRNA technology has been introduced in order to downregulate 5-ALA-synthase 1, which contributes to the patho-physiology of these diseases. Moreover, other novel therapies using enzyme protein replacement, mRNA techniques or proteostasis regulators are being developed.

Dietary Supplementation of Nile Tilapia (Oreochromis niloticus) With Panax ginseng Essential Oil: Positive Impact on Animal Health and Productive Performance, and Mitigating Effects on Atrazine- Induced Toxicity

The present study assessed the impact of Panax ginseng essential oil (GEO) supplementation on physiological parameters related to productive performance and health status in Nile tilapia reared under standard conditions and exposed to a sub-lethal atrazine (ATZ) concentration. Fish were allocated into 6 groups: the control group was reared in clean water and fed with a commercial basal diet (CNT), two groups were fed with the basal diet supplemented with two different levels of GEO (GEO1 and GEO2, respectively), one group was intoxicated with 1/5 of ATZ 96-h lethal concentration 50 (1.39 mg/L) (ATZ group), and the remaining two groups were fed with the GEO-supplemented diets and concurrently exposed to 1.39 mg ATZ/L (GEO1+ATZ and GEO2+ATZ, respectively). The experiment lasted for 60 days. GEO supplementation exerted a significantly positive influence on fish growth, feed utilization, and hepatic antioxidant defense systems at both levels of supplementation. ATZ exposure significantly reduced fish survival rates and impaired fish growth and feed utilization, with the lowest final weights, weight gain, total feed intake, and the highest feed conversion ratio being recorded in the ATZ-intoxicated group. ATZ exposure caused significant changes in intestinal digestive enzyme activity (decreased lipase activity), hematological indices (decreased hemoglobin, packed cell volume, erythrocytes, and leukocytes), blood biochemical variables (decreased total proteins, albumin, globulins, and immunoglobulin M; increased total cholesterol, triglycerides, and cortisol), and hepatic oxidative/antioxidant indices (decreased glutathione level, superoxide dismutase and catalase enzyme activity and mRNA expression levels, and increased malondialdehyde content). Moreover, in the hepatic tissue of ATZ-intoxicated Nile tilapia, histopathological alterations and upregulated mRNA expression levels of stress- and apoptosis-related genes (Hsp70, caspase 3, and p53) were observed. GEO supplementation in ATZ-treated groups significantly attenuated the aforementioned negative effects, though some parameters did not reach the CNT values. These findings provide further and partly new evidence that sub-lethal ATZ toxicity induces reduced survivability, growth retardation, impaired digestive function, anemia, immunosuppression, hepatic oxidative stress damage, and overall increased stress level in Nile tilapia, and suggest that GEO supplementation may be useful for mitigating this toxicity and provide more general support to the productive performance and health status of this fish species.

Interactive effects of high temperature and pesticide exposure on oxidative status, apoptosis, and renin expression in kidney of goldfish: Molecular and cellular mechanisms of widespread kidney damage and renin attenuation.

One of many noteworthy consequences of increasing societal reliance on pesticides is their predominance in aquatic environments. These pernicious chemicals interact with high temperatures from global climate change, heat waves, and natural variations to create unstable environments that negatively impact organisms' health. To understand these conditions, we examined the dose-dependent effects of environmentally relevant pesticide mixtures (metolachlor, linuron, isoproturon, tebuconazole, aclonifen, atrazine, pendimethalin, and azinphos-methyl) combined with elevated temperatures (22 control vs. 32°C for 4-week exposure) on renin, dinitrophenyl protein (DNP, an indicator of reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, an indicator of reactive nitrogen species, RNS), superoxidase dismutase (SOD, an antioxidant), and catalase (CAT, an antioxidant) expressions in the kidneys of goldfish (Carassius auratus). Histopathological analysis showed widespread damage to kidney tissues in high temperature and pesticide co-exposure groups, including rupture of the epithelial layer, hemorrhaging, and degeneration of tubular epithelium. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical analyses demonstrated significant declines in renin receptor-like mRNA and protein expressions in kidney tissues under combined exposure to high temperature and pesticides compared with controls; conversely, expression of DNP, NTP, SOD, and CAT increased in kidney tissues under the same conditions. Apoptotic cells were also increased in co-exposure groups as assessed by in situ terminal deoxynucleotidyl transferase dUTP nick labeling (TUNEL) assay. The enhanced apoptosis in kidneys of heat and pesticides co-exposed fish was associated with increased caspase-3 (a protease enzyme) mRNA levels. Our results demonstrated that high temperature and pesticides induced oxidative/nitrative stress (i.e., ROS/RNS), damaged tissues, increased cellular apoptosis, and suppressed renin expression in kidneys of goldfish.