Expression Of Synthetic Enzymes Research Articles

Abstract 15133: Matricellular Protein CCN1 Regulates Cardiac Fibroblast ECM Synthesis and Alignment

Introduction: Cellular communication network (CCN) proteins constitute a 6-member family of extracellular matrix (ECM)-associated signaling proteins that have important roles in wound healing and tissue fibrosis. Though their function in the heart is not fully explicated, CCN1 is often indicated as an antifibrotic factor. Yet, CCN1 levels correlate with infarct size and ventricular dysfunction in myocardial infarct patients—signifying a pro-fibrotic role for this factor in the heart. Objective: To determine the contribution of endogenous CCN1 to fibroblast (FB) ECM synthesis and organization. Methods: In vitro murine cardiac FB ECM synthesis, activation, proliferation, and matrix organization was evaluated in cells treated with CCN1 or non-target (NT) siRNA, by qPCR/immunoblotting for collagens and synthetic enzymes, EdU incorporation, and type I collagen immunostaining of decellularized matrices, respectively. CCN1-dependent integrin pathways modulating FB collagen synthetic enzyme expression were identified and interrogated via in vitro immunoprecipitation and RNAi-mediated depletion. Results: CCN1-deficient FBs showed reduced expression of type I collagen and collagen processing enzymes ( P4ha1 , P4hb , Plod1 , Plod2 , Loxl2 ). siRNA-mediated knockdown of CCN1 did not impact FB activation (α-SMA), but significantly reduced the expression of collagen synthetic/processing enzymes ( P4ha1 , Plod1 , Plod2 , Loxl2 ). Deficiency of CCN1 in both unstimulated and TGFβ1-stimulated FBs resulted in reduced proliferative capacity (7.17%, p=0.0205 and 5.34%, p=0.0428, respectively, relative to NT, n=3). CCN1 depletion promoted the alignment of FB-derived collagen fibers (0.30 ± 0.03 vs. 0.58 ± 0.04; mean ± SEM, p<0.0001, n=6). An interaction between CCN1 and integrin αvβ3 was validated; moreover, knockdown of integrin β3 reduced the expression of enzymes associated with collagen cross-linking ( Plod1 , Plod2 , Loxl2 ), partly phenocopying CCN1 depletion. Conclusions: The data suggest that CCN1 drives pro-fibrotic signaling and limits ECM organization in cardiac FBs. This may have implications for CCN1 in the infarcted heart by promoting scar establishment; however, long-term expression could lead to progressive detrimental remodeling.

Aconitine inhibits androgen synthesis enzymes by rat immature Leydig cells via down-regulating androgen synthetic enzyme expression in vitro

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0–50 μM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 μM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 μM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 μM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 μM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis.

Comparative analysis of monoaminergic cerebrospinal fluid-contacting cells in Osteichthyes (bony vertebrates).

Cerebrospinal fluid‐contacting (CSF‐c) cells containing monoamines such as dopamine (DA) and serotonin (5‐HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF‐c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5‐HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF‐c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF‐c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF‐c cells coexpress TH2 and TPH1 and contain both DA and 5‐HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF‐c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF‐c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF‐c cells, while teleosts have increased their relative number.

Melamine negatively affects testosterone synthesis in mice

Several studies have found that melamine causes damage to the testes, epididymis and sperm. However, few studies have investigated the effect of melamine on the synthesis of testosterone, which plays an import role in testicular development and spermatogenesis. In present study, mice were orally administrated with 2, 10 or 50mg/kg of melamine for 28days. In these groups, various abnormalities were observed including disruption of the seminiferous tubule structure, an increased necrotic germ cells and sperm abnormalities, and a reduced sperm count. Melamine exposure also decreased the level of serum testosterone and levels of testicular StAR, P450scc and 17β-HSD. In addition, melamine exposure reduced the number of Leydig cells. Taken together, these results indicate that melamine exposure reduces the level of testosterone through down-regulation of StAR and testosterone synthetic enzyme expression and also a decreased number of Leydig cells. This may further affect testicular development and lead to sperm damage.

The dual effect of a ferredoxin-hydrogenase fusion protein in vivo: successful divergence of the photosynthetic electron flux towards hydrogen production and elevated oxygen tolerance.

BackgroundHydrogen photo-production in green algae, catalyzed by the enzyme [FeFe]-hydrogenase (HydA), is considered a promising source of renewable clean energy. Yet, a significant increase in hydrogen production efficiency is necessary for industrial scale-up. We have previously shown that a major challenge to be resolved is the inferior competitiveness of HydA with NADPH production, catalyzed by ferredoxin-NADP+-reductase (FNR). In this work, we explored the in vivo hydrogen production efficiency of Fd-HydA, where the electron donor ferredoxin (Fd) is fused to HydA and expressed in the model organism Chlamydomonas reinhardtii.ResultsWe show that once the Fd-HydA fusion gene is expressed in micro-algal cells of C. reinhardtii, the fusion enzyme is able to intercept photosynthetic electrons and use them for efficient hydrogen production, thus supporting the previous observations made in vitro. We found that Fd-HydA has a ~4.5-fold greater photosynthetic hydrogen production rate standardized for hydrogenase amount (PHPRH) than that of the native HydA in vivo. Furthermore, we provide evidence suggesting that the fusion protein is more resistant to oxygen than the native HydA.ConclusionsThe in vivo photosynthetic activity of the Fd-HydA enzyme surpasses that of the native HydA and shows higher oxygen tolerance. Therefore, our results provide a solid platform for further engineering efforts towards efficient hydrogen production in microalgae through the expression of synthetic enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0601-3) contains supplementary material, which is available to authorized users.

Mimicking the Diabetic State in the Non‐Diabetic β‐cell to Elucidate Critical Pathways in β‐cell Dysfunction

EP3, a receptor for prostaglandin E2 (PGE2), plays a key role in the β‐cell dysfunction of diabetes. We demonstrated EP3 expression and PGE2 production are increased in islets isolated from diabetic mice and humans, and targeting this pathway in vitro restores proper β‐cell function. Yet, the mechanisms involved in the pathogenesis of β‐cell dysfunction upstream and downstream of EP3 remain elusive. The INS‐1 832/3 cell line has high EP3 expression, just as in diabetic islets. Interestingly, even though EP3 expression is high, PGE2 production is not; thus, an EP3 antagonist has no effect on β‐cell function. We hypothesized one or more of the multitude of insults β‐cells encounter in the diabetic state are necessary to stimulate PGE2 production and resulting β‐cell dysfunction. We mimicked diet by enriching INS‐1 cells with arachidonic acid (AA), an ω‐6 polyunsaturated fatty acid and PGE2 precursor, and inflammation by IL‐1β treatment. This combination significantly increased PGE2 production, blunting insulin secretion. Further, β‐cell function could be restored by an EP3 antagonist. In islets isolated from non‐diabetic mice and humans, AA and IL‐1β had a strong effect on PGE2 synthetic enzyme expression, but EP3 itself was relatively unchanged, and EP3‐mediated β‐cell dysfunction minimal. We hypothesized that high glucose or free fatty acids are responsible for the dysfunctional up‐regulation of β‐cell EP3 in vivo. Finally, we demonstrate the relevance of targeting this pathway in vivo, as mice lacking the EP3 effector, Gαz, specifically in the β‐cell are protected from the β‐cell dysfunction induced by inflammation.

CHARACTERISATION OF THE SYNTHETIC ENZYMES OF H2S IN MURINE MYOCARDIUM

Hydrogen sulphide (H2S) is a novel endogenously produced gasotransmitter. H2S donor compounds protect the heart from ischaemia-reperfusion injury. However the potential for the heart to regulate endogenous H2S availability to...

P39 Expression and localisation of H2S metabolising enzymes in the murine myocardium and liver

Background New interventions are required to prevent the loss of cardiomyocytes associated with myocardial ischemia and with the reperfusion injury that follows revascularisation. Recent studies have shown that compounds releasing hydrogen sulfide (H2S) can have cardioprotective effects [1] . However, these compounds are generally effective only at high (μM) concentrations and release is not well controlled, increasing the potential for cytotoxicity. H2S is produced endogenously and targeting the pathways that regulate availability in heart may be a more promising therapeutic approach. The present study aimed to characterise the distribution of the H2S synthesising enzymes, cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE) and mercaptopyruvate sulfurtransferase (MPST), and of H2S degrading enzyme, thiosulfate sulfurtransferase (TST) in the murine heart and in liver, an important site of H2S metabolism. Methods RNA and protein were prepared from heart and liver collected from 10 week old male C57BL/6J mice for quantitative RT-PCR and Western blotting. Further tissue was formalin fixed, processed and wax embedded before sectioning for immunohistochemistry. Results qRT-PCR showed high relative gene expression for all H2S metabolising enzymes in the liver. Cardiac tissue displayed high relative gene expression of TST & CSE, moderate expression of CBS and low expression of MPST. Western blots confirmed the presence of protein for TST & all H2S synthesising enzymes within heart & liver homogenates. Immunohistochemical staining of fixed tissue sections located CSE to vascular smooth muscle in the heart, while CBS & MPST were found in the vascular endothelium. Cardiomyocytes were immunoreactive for all enzymes. In hepatocytes, immunoreactive TST, CBS & MPST are found in hepatocytes throughout the liver, while CSE immunoreactivity is notably intensified around the central vein, responsible for hepatic venous return. Conclusion The murine myocardium and liver have the capacity to regulate local H2S availability via three separate synthetic pathways at least one degradative pathway which have distinctive cellular localisations. Future studies will investigate whether metabolic & synthetic enzyme expression and H2S availability is differentially regulated following ischaemia and reperfusion.

Corticosteroid therapy for patients in septic shock: Some progress in a difficult decision

Objectives: Reversible adrenal insufficiency has been frequently diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to adrenocorticotrophic hormone (ACTH) stimulation. It is generally accepted that a phenomenon called contributes to immunosuppression during sepsis. The present study was to investigate whether endotoxin tolerance occurs in the adrenal gland, leading to hyporesponsiveness of adrenal gland during sepsis. Design: Controlled laboratory experiment. Setting: University research laboratory. Subjects: Sprague-Dawley male rats 200-250 g, and primary isolated adrenal fasciculata-reticularis cells. Interventions: Rats received intra-arterial injection of purified lipopolysaccharide (LPS, 0.5 mg/kg) through indwelling femoral arterial catheters, and 24 h later the adrenocortical sensitivity to exogenous ACTH (10 ng/kg) was detected. Primary F/R cells were pretreated with LPS at 0.1-100 ng/mL or with ACTH at 0.01―10 ng/mL, and then challenged, in fresh media, with 1 μg/mL LPS or 10 ng/mL ACTH. Measurements and Main Results: Toll-like receptor 4 were expressed in adrenal gland and primary fasciculata-reticularis cells. Plasma corticosterone response to ACTH was decreased in rats receiving preinjection of LPS. LPS pretreatment caused a significant decrease in corticosterone production in response to subsequent ACTH and LPS stimulation in primary fasciculata-reticularis cells. LPS pretreatment inhibited ACTH- and LPS-induced expression of steroid metabolizing enzymes. LPS significantly decreased toll-like receptor 4 and ACTH receptor expression. Conclusions: Pre-exposure to LPS resulted in hyporesponsiveness to ACTH stimulation in rats. In vitro, LPS pretreatment impaired corticosterone production of F/R cells in response to ACTH and LPS, which was associated with decreased expression of synthetic enzymes required for corticosterone production. Our results indicate that endotoxin tolerance of adrenal gland is one of mechanisms for adrenocortical insufficiency during sepsis.

Endotoxin tolerance of adrenal gland: Attenuation of corticosterone production in response to lipopolysaccharide and adrenocorticotropic hormone*

Reversible adrenal insufficiency frequently has been diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to adrenocorticotropic hormone (ACTH) stimulation. It is generally accepted that a phenomenon called "endotoxin tolerance" contributes to immunosuppression during sepsis. The present study was to investigate whether endotoxin tolerance occurs in the adrenal gland, leading to hyporesponsiveness of adrenal gland during sepsis. Controlled laboratory experiment. University research laboratory. Sprague-Dawley male rats 200-250 g and primary isolated adrenal fasciculata-reticularis cells. Rats received intra-arterial injection of purified lipopolysaccharide (0.5 mg/kg) through indwelling femoral arterial catheters, and 24 hrs later the adrenocortical sensitivity to exogenous ACTH (10 ng/kg) was detected. Primary fasciculata-reticularis cells were pretreated with lipopolysaccharide at 0.1-100 ng/mL or with ACTH at 0.01-10 ng/mL and then challenged, in fresh media, with 1 μg/mL lipopolysaccharide or 10 ng/mL ACTH. Toll-like receptor 4 was expressed in adrenal gland and primary fasciculata-reticularis cells. Plasma corticosterone response to ACTH was decreased in rats receiving preinjection of lipopolysaccharide. Lipopolysaccharide pretreatment caused a significant decrease in corticosterone production in response to subsequent ACTH and lipopolysaccharide stimulation in primary fasciculata-reticularis cells. Lipopolysaccharide pretreatment inhibited ACTH- and lipopolysaccharide-induced expression of steroid metabolizing enzymes. Lipopolysaccharide significantly decreased Toll-like receptor 4 and ACTH receptor expression. Pre-exposure to lipopolysaccharide resulted in hyporesponsiveness to ACTH stimulation in rats. In vitro, lipopolysaccharide pretreatment impaired corticosterone production of fasciculata-reticularis cells in response to ACTH and lipopolysaccharide, which was associated with decreased expression of synthetic enzymes required for corticosterone production. Our results indicate that endotoxin tolerance of adrenal gland is one of the mechanisms for adrenocortical insufficiency during sepsis.

Induction of avian musculoaponeurotic fibrosarcoma proteins by toxic bile acid inhibits expression of glutathione synthetic enzymes and contributes to cholestatic liver injury in mice

We previously showed that hepatic expression of glutathione (GSH) synthetic enzymes and GSH levels fell 2 weeks after bile duct ligation (BDL) in mice. This correlated with a switch in nuclear anti-oxidant response element (ARE) binding activity from nuclear factor erythroid 2-related factor 2 (Nrf2) to c-avian musculoaponeurotic fibrosarcoma (c-Maf)/V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (MafG). Our current aims were to examine whether the switch in ARE binding activity from Nrf2 to Mafs is responsible for decreased expression of GSH synthetic enzymes and the outcome of blocking this switch. Huh7 cells treated with lithocholic acid (LCA) exhibited a similar pattern of change in GSH synthetic enzyme expression as BDL mice. Nuclear protein levels of Nrf2 fell at 20 hours after LCA treatment, whereas c-Maf and MafG remained persistently induced. These changes translated to ARE nuclear binding activity. Knockdown of c-Maf or MafG individually blunted the LCA-induced decrease in Nrf2 ARE binding and increased ARE-dependent promoter activity, whereas combined knockdown was more effective. Knockdown of c-Maf or MafG individually increased the expression of GSH synthetic enzymes and raised GSH levels, and combined knockdown exerted an additive effect. Ursodeoxycholic acid (UDCA) or S-adenosylmethionine (SAMe) prevented the LCA-induced decrease in expression of GSH synthetic enzymes and promoter activity and prevented the increase in MafG and c-Maf levels. In vivo knockdown of the Maf genes protected against the decrease in GSH enzyme expression, GSH level, and liver injury after BDL. Toxic bile acid induces a switch from Nrf2 to c-Maf/MafG ARE nuclear binding, which leads to decreased expression of GSH synthetic enzymes and GSH levels and contributes to liver injury during BDL. UDCA and SAMe treatment targets this switch.

NO and CO have got to GO for enhanced chemoreceptor sympathoexcitation in heart failure

arterial chemoreceptors located in the carotid body are an important defense mechanism against systemic hypoxemia. Activation of arterial chemoreceptors increases alveolar ventilation and sympathetic outflow to most vascular beds. During acute exposures to hypoxia, the sympathoexcitatory responses

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.