Constitutive Synthesis Research Articles

Inhibiting Triggering Receptor Expressed on Myeloid Cells-1 signaling to ameliorate skin fibrosis.

Systemic sclerosis (SSc) is characterized by immune system failure, vascular insult, autoimmunity, and tissue fibrosis. Transforming growth factor-beta (TGF-β) is a crucial mediator of persistent myofibroblast activation and aberrant extracellular matrix production in SSc. The factors responsible for this are unknown. By amplifying pattern recognition receptor signaling, Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) is implicated in multiple inflammatory conditions. In this study, we used novel ligand-independent TREM-1 inhibitors in order to investigate the pathogenic role of TREM-1 in SSc, using preclinical models of fibrosis, and explanted SSc skin fibroblasts. Selective pharmacological TREM-1 blockade prevented and reversed skin fibrosis induced by bleomycin in mice and mitigated constitutive collagen synthesis and myofibroblast features in SSc fibroblasts in vitro. Our results implicate aberrantly activated TREM-1 signaling in SSc pathogenesis, identify a unique approach to TREM-1 blockade, and suggest a potential therapeutic benefit for TREM-1 inhibition.

Turning the industrially relevant marine alga Nannochloropsis red: one move for multifaceted benefits.

Nannochloropsis oceanica is an industrially relevant marine microalga rich in eicosapentaenoic acid (EPA, a valuable ω-3 polyunsaturated fatty acid), yet the algal production potential remains to be unlocked. Here we engineered N. oceanica to synthesize the high-value carotenoid astaxanthin independent of high-light (HL) induction for achieving multifaceted benefits. By screening β-carotenoid ketolases and hydroxylases of various origins, and strategically manipulating compartmentalization, fusion patterns, and linkers of the enzyme pair, a remarkable 133-fold increase in astaxanthin content was achieved in N. oceanica. Iterative metabolic engineering efforts led to further increases in astaxanthin synthesis up to 7.3 mg g-1, the highest reported for microalgae under nonstress conditions. Astaxanthin was found in the photosystem components and allowed the alga HL resistance and augmented EPA production. Besides, we achieved co-production of astaxanthin and EPA by the engineered alga through a fed-batch cultivation approach. Our findings unveil the untapped potential of N. oceanica as a robust, light-driven chassis for constitutive astaxanthin synthesis and provide feasible strategies for the concurrent production of multiple high-value biochemicals from CO2, thereby paving the way for sustainable biotechnological applications of this alga.

Genetically engineered Lactococcus lactis strain constitutively expresses GABA-producing genes and produces high levels of GABA.

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter of the central nervous system that impacts physical and mental health. Low GABA levels have been documented in several diseases, including multiple sclerosis and depression, and studies suggest that GABA could improve disease outcomes in those conditions. Probiotic bacteria naturally produce GABA and have been engineered to enhance its synthesis. Strains engineered thus far use inducible expression systems that require the addition of exogenous molecules, which complicates their development as therapeutics. This study aimed to overcome this challenge by engineering Lactococcus lactis with a constitutive GABA synthesis gene cassette. GABA synthesizing and transport genes (gadB and gadC) were cloned onto plasmids downstream of constitutive L. lactis promoters [P2, P5, shortened P8 (P8s)] of different strengths and transformed into L. lactis. Fold increase in gadCB expression conferred by these promoters (P2, P5, and P8s) was 322, 422, and 627, respectively, compared to the unmodified strain (P=0.0325, P8s). GABA synthesis in the highest gadCB expressing strain, L. lactis-P8s-glutamic acid decarboxylase (GAD), was dependent on media supplementation with glutamic acid and significantly higher than the unmodified strain (P<0.0001, 125mM, 200mM glutamic acid). Lactococcus lactis-P8s-GAD is poised for therapeutic testing in animal models of low-GABA-associated disease.

Ранні маркери гострої доксорубіциніндукованої кардіотоксичності та механізми її розвитку

Arrhythmias and congestive heart failure, which pose the greatest risk of toxic cardiomyopathy, are the clinically limiting side effects of doxorubicin, the main highly active anticancer agent. The difficulty of early diagnosis of cardiomyopathy and timely detection of cardiac dysfunction associated with chemotherapy remains a significant medical problem. The aim of our study was to identify early signs of acute doxorubicin-induced cardiotoxicity in adult rats by assessing ECG changes and biochemical parameters. Acute cardiotoxicity was modelled by short-term intraperitoneal injection of doxorubicin at a total dose of 15 mg/kg. On the 5th day of the experiment, visual fluctuations of electrocardiogram (ECG) waveforms, duration and amplitude of the main teeth and intervals, as well as heart rate (HR) in the control and experimental groups of rats were studied to determine early ECG signs of cardiotoxicity. The most significant ECG changes were a doubling of the QT interval duration and significant ST-segment elevation in the rats of the experimental group. In experiments on isolated aortic rings, we demonstrated doxorubicin-induced disruption of both vascular relaxation and contraction mechanisms. The endothelium-dependent relaxation of vascular preparations of animals after administration of doxorubicin to acetylcholine (0.1 μmol/l) was 47% less than in the control group. The vascular ring contractions in rats under the influence of norepinephrine (10 μmol/l) were 59% lower than in control rats. After doxorubicin administration, oxidative stress developed against the background of cardiovascular disorders. Thus, the content of diene conjugates and malondialdehyde increased by 4 and 2.5 times, respectively. At the same time, in isolated cardiac mitochondria, the activity of inducible NO synthase increased 3.7-fold with a simultaneous significant 4.8-fold inhibition of constitutive NO synthase. An increase in the content of acute-phase biochemical parameters that are markers of damage, namely alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine phosphokinase myocardial fraction (CPK MB) by 2.2, 1.4 and 1.5 times, respectively, was detected. Thus, the acute cardiotoxic effect of doxorubicin results in changes in the systolic-diastolic function of the left ventricle of the heart and its conduction, automaticity and contractility, as well as impaired relaxation and contraction of isolated preparations of the aorta against the background of increased activity of inducible and decreased constitutive NO synthesis, accompanied by oxidative stress and increased content of biochemical markers of myocardial damage.

A phenomenological model of non-genomic variability of luminescent bacterial cells

The light emitted by a luminescent bacterium serves as a unique native channel of information regarding the intracellular processes within the individual cell. In the presence of highly sensitive equipment, it is possible to obtain the distribution of bacterial culture cells by the intensity of light emission, which correlates with the amount of luciferase in the cells. When growing on rich media, the luminescence intensity of individual cells of brightly luminous strains of the luminescent bacteria Photobacterium leiognathi and Ph. phosporeum reaches 104–105 quanta/s. The signal of such intensity can be registered using sensitive photometric equipment. All experiments were carried out with bacterial clones (genetically homogeneous populations). A typical dynamics of luminous bacterial cells distributions with respect to intensity of light emission at various stages of batch culture growth in a liquid medium was obtained. To describe experimental distributions, a phenomenological model that links the light of a bacterial cell with the history of events at the molecular level was constructed. The proposed phenomenological model with a minimum number of fitting parameters (1.5) provides a satisfactory description of the complex process of formation of cell distributions by luminescence intensity at different stages of bacterial culture growth. This may be an indication that the structure of the model describes some essential processes of the real system. Since in the process of division all cells go through the stage of release of all regulatory molecules from the DNA molecule, the resulting distributions can be attributed not only to luciferase, but also to other proteins of constitutive (and not only) synthesis.

Ecdysterone treatment restores constitutive NO synthesis and alleviates oxidative damage in heart tissue and mitochondria of streptozotocin-induced diabetic rats

Constitutive NO synthases (cNOS) are the primary targets of diabetes mellitus and the impairment of cNOS functioning in cardiovascular system is one of the hallmarks of this disease. The aim of this work was to study the effect of a plant sterol ecdysterone (20-β-hydroxyecdysterone) on the NO synthases functioning and RNS metabolism in heart mitochondria and the heart tissue in the rat model of streptozotocin-induced type I diabetes. Diabetes development resulted in cNOS dysfunction both in heart mitochondria and heart tissue. cNOS activity was dramatically suppressed, but 3-fold and 6-fold rise of iNOS activity was observed in mitochondria and heart tissue respectively. Also, in mitochondria there was ~2.5 time’s increase in urea content and the activity of arginase 2 (ARG2), which could compete with NOS for the common substrate L-arginine. Total RNS production was dramatically elevated in mitochondria of diabetic animals, which well agreed with iNOS activation. Unlike this, in heart tissue dramatic increase of iNOS activity increased the content of nitrosothiols (RSNO), while total RNS production remained close to control. Both in the heart tissue and mitochondria, there was dramatic augmentation of superoxide production that correlated with sharp elevation of iNOS activity and steep rise of diene conjugates (DC) content, which indicated strong lipid oxidation. Ecdysterone treatment resulted in the reduction of iNOS activity and twofold elevation of mtNOS activity as compared to control. However, in the whole heart tissue eNOS was restored only by half of control level, which indicated specific action of ecdysterone on mtNOS isoform. RNS production returned to control in mitochondria, and was by half reduced in the heart tissue, which indicated the abolition of nitrosative stress. Correlation dependence between iNOS activity and superoxide production was found in mitochondria, which could indicate iNOS uncoupling. The restoration of cNOS activity and the reduction of iNOS activity to control level after ecdysterone treatment well correlated with the reduction of superoxide production and indicated possible ‘iNOS re-coupling‘, which resulted in the reduction of DC formation to control level. So, STZ-induced type I diabetes dramatically up-regulated iNOS activity and suppressed cNOS activity. Ecdysterone treatment reduced iNOS activity and restored constitutive NO biosynthesis to control level, which abolished oxidative and nitrosative stress in cardiac mitochondria and heart tissue of STZ-induced diabetic animals. Possible pathways involved in ecdysterone action on constitutive NO biosynthesis were discussed.

Fatty acid composition and metabolic partitioning of α-linolenic acid are contingent on life stage in human CD3+ T lymphocytes.

Immune function changes across the life course; the fetal immune system is characterised by tolerance while that of seniors is less able to respond effectively to antigens and is more pro-inflammatory than in younger adults. Lipids are involved centrally in immune function but there is limited information about how T cell lipid metabolism changes during the life course. We investigated whether life stage alters fatty acid composition, lipid droplet content and α-linolenic acid (18:3ω-3) metabolism in human fetal CD3+ T lymphocytes and in CD3+ T lymphocytes from adults (median 41 years) and seniors (median 70 years). Quiescent fetal T cells had higher saturated (SFA), monounsaturated fatty acid (MUFA), and ω-6 polyunsaturated fatty acid (PUFA) contents than adults or seniors. Activation-induced changes in fatty acid composition differed between life stages. The principal metabolic fates of [13C]18:3ω-3 were constitutive hydroxyoctadecatrienoic acid synthesis and β-oxidation and carbon recycling into SFA and MUFA. These processes declined progressively across the life course. Longer chain ω-3 PUFA synthesis was a relatively minor metabolic fate of 18:3ω-3 at all life stages. Fetal and adult T lymphocytes had similar lipid droplet contents, which were lower than in T cells from seniors. Variation in the lipid droplet content of adult T cells accounted for 62% of the variation in mitogen-induced CD69 expression, but there was no significant relationship in fetal cells or lymphocytes from seniors. Together these findings show that fatty acid metabolism in human T lymphocytes changes across the life course in a manner that may facilitate the adaptation of immune function to different life stages.

EFFECT OF TRANSCRIPTION FACTOR MODULATORS ON OXIDATIVE-NITROSATIVE STRESS INDICATORS IN TISSUES OF THE SMALL INTESTINE OF RATS AFTER LAPARATOMY UNDER LIPOPOLYSACCHARIDE-INDUCED SYSTEMIC INFLAMMATORY RESPONSE

This study is devoted to investigating the effect of NF-κB and Nrf2 transcription factor modulators on oxidative-nitrosative stress indicators in the tissues of the small intestine of rats after abdominal surgical trauma (laparotomy) under lipopolysaccharide (LPS)-induced systemic inflammatory response (SIR). The study was performed on 35 Wister white male rats weighing 220-250 g, divided into 5 groups: the 1st (control) group included "sham-operated" animals; the 2nd group involved rats, who underwent laparotomy following the LPS-induced SIR modelling; the animals of the 3rd, 4th and 5th groups subjected to laparotomy following LPS-induced SIR and then for 7 days received ammonium pyrrolidine dithiocarbamate, an NF-κB activation inhibitor, in a dose of 76 mg/kg intraperitoneally, dimethyl fumarate, an Nrf2 inducer, in a dose of 15 mg/kg in 10% dimethyl sulfoxide solution, and a water-soluble form of quercetin in a dose of 100 mg/kg that is 10 mg/kg in terms of quercetin, respectively. The measurements were carried out in 7 days after the “sham” surgical operation and laparotomy. It has been found out that the use of ammonium pyrrolidine dithiocarbamate, dimethyl fumarate, and water-soluble form of quercetin in case of the combined effects of surgical trauma (laparotomy) and LPS S. typhi significantly restrains the production of superoxide anion radical by various sources (microsomes and constitutive isoforms of NO-synthase, leukocyte NADPH-oxidase), total and inducible NO-synthase activity, formation of peroxynitrite in the tissues of the small intestine of rats. The study has also demonstrated the ability of dimethylfumarate, unlike ammonium pyrrolidine dithiocarbamate and quercetin, to improve the constitutive synthesis of nitric oxide under experimental conditions.

The Rhizobium tropici CIAT 899 NodD2 protein promotes symbiosis and extends rhizobial nodulation range by constitutive nodulation factor synthesis.

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specific nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the five NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as efficiently as the wild type. Interestingly, CIAT 899 NodD2-mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fixing nodules in two host legumes but also results in nodule development in incompatible legumes. These findings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.

Stimulation of the endogenous hydrogen sulfide synthesis restores constitutive nitric oxide synthesis and endothelium-dependent vasodilation in old rats

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Hydrogen sulfide (H2S) is a gas transmitter of endogenous origin, which has an important regulatory function in the human body. Endogenous H2S is synthesized by three enzymes, cistationine-β-synthase (CBS), cistationine-γ-liase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MTS), which is coupled to the activity of cysteine aminotransferase (CAT). CAT, CBS, and CSE are pyridoxal-5- phosphate (PLP)-dependent enzymes. It is known that with age the exchange of hydrogen sulfide is disturbed against the background of increasing oxidative and nitrosative stress. At the same time there is a decrease of constitutive synthesis of nitric oxide (NO). Recent studies show that hydrogen sulfide closely interacts with the NO system. Activation of H2S biosynthesis and restoration of its pools in tissues of old animals may be a promising direction of cardiovascular disease prevention. The aim of the current work was to study the effect of H2S-synthesizing enzymes cofactor pyridoxal-5-phosphate (PLP) on the endothelium-dependent relaxation of smooth muscle vessels in old rats, as well as the H2S content and cNOS activity in aortic tissues. Methods Old rats (22-24 months old) were PLP administered per os in dose of 0,7 mg per kg daily for 2 weeks.The content of hydrogen sulfide, activity of cNOS and iNOS was measured in aortic tissues.Vascular reactions were determined in experiments on isolated aortic strips. To clarify the role of either NO or H2S in the endothelium-dependent vasorelaxation, N-nitro-L-arginine methyl ester hydrochloride (L-NAME) (Sigma, USA, 27 mg/kg) or O-carboxymethylhydroxylamine (O-CMH) (50 mg/kg) (Sigma, USA) was injected intraperitoneally 30 min before sacrificing. Results We showed that the H2S content was 1.6 times lower in old rat aorta compared to adult ones. PLP administration for 2 weeks induced a significant increase of H2S content in aortic tissue of old rats (P&amp;lt;0.05). It was found that with aging, the activity of cNOS decreases by 3 times, and the stimulation of endogenous Н2S leads to an increase in the activity of this enzyme in the aortic tissues by 2.1 times as compared with those in old animals. At the same time, the activity of the iNOS enzyme with aging increases by 3.8 times, and after the introduction of PLP, it decreases almost halved. Also shown, that PLP significantly improved endothelium-dependent aortic smooth muscle relaxation in old rats. The amplitude of acetylcholine-induced relaxation which was significantly reduced in old rats was increased more than 2.5-fold. This effect was reversed by inhibitors of NO-synthase and 3-mercaptopyruvate sulfurtransferase indicating the involvement of NO and H2S in the improved endothelium-dependent vascular relaxation. Conclusions Thus, H2S acts as a regulator of the NO system that increases cNOS activity and NO synthesis in aortic tissue, which improves endothelium-dependent relaxation of aortic smooth muscle in aged rats.

Dual regulation of lipid droplet-triacylglycerol metabolism and ERG9 expression for improved \u03b2-carotene production in Saccharomyces cerevisiae

BackgroundThe limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve β-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches.ResultsThe direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of β-carotene in S. cerevisiae. However, due to the competition for precursor between β-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on β-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in β-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to β-carotene pathway and achieved additional 31.7% increase in β-carotene content without adversely affecting cell growth. By inducing an extra constitutive β-carotene synthesis pathway for further conversion precursor farnesol to β-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of β-carotene, which is 107.3% and 49.5% increase respectively over the parent strain.ConclusionsThis strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae.Graphical

COMPARATIVE CHARACTERISTICS OF HUMAN INTERFERON-GAMMA PREPARATIONS FROM DIFFERENT PRODUCER STRAINS

Here, we performed a comparative evaluation of the effectiveness of the isolation of human interferon-gamma from the biomass of the recombinant strain of E.coli MH-1/pIFN-γ-trp2 enabling constitutive synthesis of the target protein in the "inclusion bodies", and a new recombinant inducible strain of E. coli BL 21/pET-IFN-γ containing the protein in a soluble form. It has been shown that the use of an inducible producer strain can increase the yield of the target protein from 1.5 mg/g to 5 mg/g and enhance the specific (antiviral) activity by 20-fold.

Pyridoxal-5-phosphate induced cardioprotection in aging associated with up-expression of cystathionine-γ-lyase, 3-mercaptopyruvate sulfurtransferase, and ATP-sensitive potassium channels.

In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). PLP was administered per os in a dose of 0.7mg per kg daily for 2weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids.

The high-value carotenoid astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type–like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid.

Комбіноване застосування пропалгілгліцину та L-цистеїну попереджає оксидативний та нітрозативний стрес у тканинах серця при фокальній ішемії-реперфузії головного мозку

Cerebral ischemia is a neurogenerative disoder that leads to partial or general paralysis and subsequent disability. The development of oxidative-nitrosative stress on the background of insufficient production of nitric oxide (NO) and hydrogen sulfide (H2S) are the main reasons behind the pathogenesis of focal ischemia-reperfusion and cerebrocardial syndrome. We studied the combined use of propargylglycine and L-cysteine as drugs that prevent oxidative and nitrosative stress and are activators of gasotransmitters - NO and H2S in the heart tissues of rats with focal ischemia. It was shown that focal ischemiareperfusion was accompanied by a significant increase in the heart of rats calcium-independent inducible synthesis of NO (iNOS) and an increase in markers of oxidative stress (superoxide anion radical, hydroxyl radical, diene conjugates) NO-synthase (cNOS). This caused disruption of nitric oxide synthesis due to the uncoupling state of cNOS in the rat heart. The use of a combination of DL-proparlgylglycine (11.31 mg/ kg) and L-cysteine (112.1 mg / kg) 40 min before the modeling of focal ischemia significantly reduced the activity of iNOS and the content of markers of oxidative metabolism in the heart of adult rats and increased the constitutive synthesis of NO, which led to restoration of the cNOS incoupling. We observed activation of endogenous synthesis of H2S, which interacts closely with the nitric oxide system and is a powerful antioxidant. It should also be noted an increase in animal survival after 24 h by 25%. Thus, the combined use of propargylglycine and Lcysteine in rats prevented disruption of NO and H2S synthesis in cardiac tissues in ishemia-reperfusion due to a slowing of the development of oxidative stress, which helped to restore cNOS coupling.

Dissociation of functional and structural plasticity of dendritic spines during NMDAR and mGluR-dependent long-term synaptic depression in wild-type and fragile X model mice

Many neurodevelopmental disorders are characterized by impaired functional synaptic plasticity and abnormal dendritic spine morphology, but little is known about how these are related. Previous work in the Fmr1-/y mouse model of fragile X (FX) suggests that increased constitutive dendritic protein synthesis yields exaggerated mGluR5-dependent long-term synaptic depression (LTD) in area CA1 of the hippocampus, but an effect on spine structural plasticity remains to be determined. In the current study, we used simultaneous electrophysiology and time-lapse two photon imaging to examine how spines change their structure during LTD induced by activation of mGluRs or NMDA receptors (NMDARs), and how this plasticity is altered in Fmr1-/y mice. We were surprised to find that mGluR activation causes LTD and AMPA receptor internalization, but no spine shrinkage in either wildtype or Fmr1-/y mice. In contrast, NMDAR activation caused spine shrinkage as well as LTD in both genotypes. Spine shrinkage was initiated by non-ionotropic (metabotropic) signaling through NMDARs, and in wild-type mice this structural plasticity required activation of mTORC1 and new protein synthesis. In striking contrast, NMDA-induced spine plasticity in Fmr1-/y mice was no longer dependent on acute activation of mTORC1 or de novo protein synthesis. These findings reveal that the structural consequences of mGluR and metabotropic NMDAR activation differ, and that a brake on spine structural plasticity, normally provided by mTORC1 regulation of protein synthesis, is absent in FX. Increased constitutive protein synthesis in FX appears to modify functional and structural plasticity induced through different glutamate receptors.

I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

A Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activitymannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.

Attenuation of chronic antiviral T-cell responses through constitutive COX2-dependent prostanoid synthesis by lymph node fibroblasts.

Lymphoid T-zone fibroblastic reticular cells (FRCs) actively promote T-cell trafficking, homeostasis, and expansion but can also attenuate excessive T-cell responses via inducible nitric oxide (NO) and constitutive prostanoid release. It remains unclear how these FRC-derived mediators dampen T-cell responses and whether this occurs in vivo. Here, we confirm that murine lymph node (LN) FRCs produce prostaglandin E2 (PGE2) in a cyclooxygenase-2 (COX2)-dependent and inflammation-independent fashion. We show that this COX2/PGE2 pathway is active during both strong and weak T-cell responses, in contrast to NO, which only comes into play during strong T-cell responses. During chronic infections in vivo, PGE2-receptor signaling in virus-specific cluster of differentiation (CD)8 cytotoxic T cells was shown by others to suppress T-cell survival and function. Using COX2flox/flox mice crossed to mice expressing Cre recombinase expression under control of the CC chemokine ligand (CCL19) promoter (CCL19cre), we now identify CCL19+ FRC as the critical source of this COX2-dependent suppressive factor, suggesting PGE2-expressing FRCs within lymphoid tissues are an interesting therapeutic target to improve T-cell–mediated pathogen control during chronic infection.

In vivo metal selectivity of metal-dependent biosynthesis of cobalt-type nitrile hydratase in Rhodococcus bacteria: a new look at the nitrile hydratase maturation mechanism?

This study highlights the effect of heavy metal ions on the expression of cobalt-containing nitrile hydratase (NHase) in Rhodococcus strains, which over-produce this enzyme. Both metal-dependent derepression of transcription and maturation of NHase were considered. We demonstrated that nickel ions can derepress the NHase promoter in several Rhodococcus strains. The cblA gene of a cobalt-dependent transcriptional repressor was shown to be indispensable for nickel-mediated derepression. As for maturation, we showed that nickel ions could not replace cobalt ions during the synthesis of active NHase. We also revealed that the amount of β-subunit decreased during NHase expression without added cobalt. We showed this using three variants of NHase in vivo synthesis: by using nickel- or urea-induced synthesis in cblA+ strains, and by using metal-independent constitutive synthesis in cblA- strains. In all cases, we found that the amount of β-subunit was significantly lower than the amount of α-subunit. In contrast, equimolar amounts of both subunits were synthesized after growth in the presence of added cobalt. Nickel did not affect NHase synthesis in mixtures with cobalt. This suggests that the metal selectivity in cblA-dependent regulation of NHase transcription was too low to discriminate between cobalt and nickel, but the selectivity of the NHase maturation mechanism was high enough to do so. Moreover, we can assume that the β-subunit is more subject to proteolytic degradation without the addition of cobalt, than the α-subunit. This indicates that cobalt ions presumably play an unknown role in the stability of the β-subunit in vivo.