Role of Nerve Growth Factor in Oxidanl Homeostasis: Glutathione Metabolism

Abstract— Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin-like NGF-responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased γ-glutamylcysteine synthetase (GCS) activity, the rate-limiting enzyme for GSH synthesis, by 50% after 9h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6-phosphate dehydroge-nase by 50% after 2 days. Treatment with NGF and cyclo-heximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6-phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide-induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 μMl -buthionine-(S,R)-sulfoximine, an inhibitor of GCS. However, treatment with 10 μMl -buthionine-(S,R)-sulfoximine alone did not affect PC12 cell viability, NGF stimulation of neurite extension, and NGF induction of GCS, GSH peroxidase, and glucose 6-phosphate dehydrogenase activity. When GSH levels were measured in PC12 cells that were treated for 24 h with other neurotrophins and growth factors, such as brain-derived neurotrophic factor, neurotro-phin-3, epidermal growth factor, insulin-like growth factor-I, and basic fibroblast growth factor, only epidermal growth factor was found to increase GSH levels by 30%. Whereas NGF increased GSH levels in the human neuro-blastoma SK-N-SH-SY5Y and the human melanoma A-875 in serum-free medium, addition of fetal calf serum to the medium abolished the NGF effects on GSH levels in the NGF-responsive cell lines, SK-N-SH-SY5Y, A-875, and the CNS C6 rat glioma subclone 2BD.     

Deubiquitinases Maintain Protein Homeostasis and Survival of Cancer Cells upon Glutathione Depletion

1. Download : Download high-res image (149KB)
2. Download : Download full-size image

     

Cell Growth and Size Homeostasis in Silico

Cell growth in size is a complex process coordinated by intrinsic and environmental signals. In a research work performed by a different group, size distributions of an exponentially growing population of mammalian cells were used to infer cell-growth rate in size. The results suggested that cell growth was neither linear nor exponential, but subject to size-dependent regulation. To explain the observed growth pattern, we built a mathematical model in which growth rate was regulated by the relative amount of mRNA and ribosomes in a cell. Under the growth model and a stochastic division rule, we simulated the evolution of a population of cells. Both the sampled growth rate and size distribution from this in silico population agreed well with experimental data. To explore the model space, alternative growth models and division rules were studied. This work may serve as a starting point to understand the mechanisms behind cell growth and size regulation using predictive models.     

Cell Growth and Size Homeostasis in Silico

Cell growth in size is a complex process coordinated by intrinsic and environmental signals. In a research work performed by a different group, size distributions of an exponentially growing population of mammalian cells were used to infer cell-growth rate in size. The results suggested that cell growth was neither linear nor exponential, but subject to size-dependent regulation. To explain the observed growth pattern, we built a mathematical model in which growth rate was regulated by the relative amount of mRNA and ribosomes in a cell. Under the growth model and a stochastic division rule, we simulated the evolution of a population of cells. Both the sampled growth rate and size distribution from this in silico population agreed well with experimental data. To explore the model space, alternative growth models and division rules were studied. This work may serve as a starting point to understand the mechanisms behind cell growth and size regulation using predictive models.     

Alteration in Glutathione Homeostasis and Oxidative Stress During the Sequelae of Trimethyltin Syndrome in Rat Brain

Trimethyltin (TMT), a by-product of tin, is used in a wide variety of industrial and agricultural purposes which serves as a model neurotoxicant in hippocampal neurodegeneration, and this could, in turn, be exploited for various therapeutic compounds essential for hippocampal neurodegeneration. Therefore, the present investigation explores the sequential changes in behavior, oxidative burden, and apoptosis following TMT administration in rat hippocampus. Male SD rats weighing 250 g were given single dose of 8.5 mg/kg TMT (i.p.) that resulted in “TMT syndrome” which begins at the third post-TMT exposure and continued till 21 days posttreatment. This resulted in behavioral alteration (aggression and spontaneous seizures), cognitive impairment as assessed by plus maze, and passive avoidance resulting in short-term memory deficits. These behavioral alterations were associated with an increase in oxidative stress. The levels of malondialdehyde, reactive oxygen species, and protein carbonyl were significantly increased (p?<?0.001) in the TMT-treated rats after the third day of exposure and were maximum at day 14 postexposure. The glutathione system was not able to adapt rapidly in response to oxidative stress which resulted in imbalance in redox status. The imbalance in the redox state resulted in the death of neurons as seen by a significant increase in caspase activation at gene as well as protein level after TMT exposure on day 14, quoting an extent of changes. Therefore, it is proposed that behavioral deficits could be accounted by the impairment of endogenous glutathione homeostasis which resulted in death of neurons in the hippocampal region.     

MoTea4-Mediated Polarized Growth Is Essential for Proper Asexual Development and Pathogenesis in Magnaporthe oryzae

Polarized growth is essential for cellular development and function and requires coordinated organization of the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly and bipolar growth in fission yeast and directional mycelial growth in Aspergillus. Here, we characterize Tea4 in the rice blast fungus Magnaporthe oryzae (MoTea4). Similar to its orthologs, MoTea4-green fluorescent protein (MoTea4-GFP) showed punctate distribution confined to growth zones, particularly in the mycelial tips, aerial hyphae, conidiophores, conidia, and infection structures (appressoria) in Magnaporthe. MoTea4 was dispensable for vegetative growth in Magnaporthe. However, loss of MoTea4 led to a zigzag morphology in the aerial hyphae and a huge reduction in conidiation. The majority of the tea4Δ conidia were two celled, as opposed to the tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coil domains of MoTea4 are necessary for proper conidiation in Magnaporthe. The tea4Δ conidia failed to produce proper appressoria and consequently failed to infect the host plants. The tea4Δ conidia and germ tubes showed disorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to the wild-type conidia, the tea4Δ conidia showed aberrant germination, poor cytoplasmic streaming, and persistent accumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment of germinating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorial development in Magnaporthe. We show that MoTea4 plays an important role in organizing the F-actin cytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and pathogenic development in Magnaporthe.Both unicellular and multicellular organisms have the ability to dynamically reorganize their cytoskeletons in response to environmental changes, as well as during polarized growth that is crucial for proliferation, differentiation, and morphogenesis. Fungal cells represent a perfect example of polarized growth that efficiently responds to environmental cues. The induction of cell polarity is particularly dramatic in fungi that show dimorphic growth or morphogenic transition and is often associated with virulence in the pathogenic species (9).Magnaporthe oryzae, a filamentous ascomycete and the causal agent of cereal blast disease, undergoes vegetative or infectious growth in the presence of rich nutrients or a host inductive surface, respectively. Under such growth conditions, Magnaporthe shows unipolar extension of the mycelial tips (or germ tubes), and its growth is well coordinated with morphogenic differentiation only under asexual or pathogenic development. Polarized growth is induced at several points in M. oryzae during the infection cycle: emergence of a germ tube from the conidium, elongation of the germ tube, penetration peg formation by the appressorium, both intra- and intercellular extension of the invasive hyphae, and development of aerial hyphae from the mass of invasive hyphae within the host. The aerial hypha starts to swell at the tip, suggestive of conidiophore initiation, which includes the hypha (conidiophore stalk) and the swollen tip (conidiophore vesicle), separated by a septum at the neck. The vesicle eventually develops into a mature 3-celled conidium (6). In yeasts, polarized growth is regulated by several proteins in a polarisome complex at the growth zone, which in turn depends on polarity pathways that are temporally and spatially regulated (12). Therefore, proteins that control morphogenic differentiation through cell polarity might play key roles in microbial pathogenesis and in adaptation to new environments.Members of the Rho family of small GTP-binding proteins act as pivotal signaling switches and play a key role in morphogenesis during pathogenic development in M. oryzae (28, 29). In Aspergillus nidulans, cell end marker proteins TeaA (Tea1), TeaR (Mod5), KipA (Tea2), and TeaC (Tea4) have been shown to be important for polarized hyphal growth (10, 24). Similarly, the Tea1 homolog ClaKel2 has been implicated in pathogenic differentiation in Colletotrichum lagenarium (18). It has been reported that a cyclin-dependent kinase from the Cdk5/Pho85 family plays a key role in regulating polar growth required for developing infection structure and virulence in the dimorphic fungal pathogen Ustilago maydis (4).Other polarity factors, apart from Tea1 and Tea2, include Tea3, Tea4, Tip1, Pom1, and Bud6 (21, 22, 26). Tea1 localizes to the cell tips in a Mod5-dependent manner (21) and is required for the recruitment of Pom1 kinase (2, 25), Bud6 (8), and the formin For3, which nucleates F-actin in Schizosaccharomyces pombe (7). Importantly, Tea4 mediates the interaction between Tea1 and For3, which is essential for F-actin nucleation in S. pombe (13). However, the role of Tea4 has not been defined in pathogenic fungi that undergo morphogenic differentiation in response to cues from the host or the environment.In this study, we show that Tea4 in M. oryzae (MoTea4) plays an important role in maintaining polarized growth of aerial hyphae during asexual development and in differentiation of germ tubes into appressoria during pathogenic growth. We analyze the importance of microtubule and actin cytoskeletal organization in appressorial development and the effect of the loss of Tea4 function on the organization of the actin cytoskeleton in M. oryzae. Lastly, MoTea4 function was found to be important for asexual differentiation and effective virulence in M. oryzae.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Linearized Growth Curve,Proper Age and Age at Menarche

A mathematical model of the linearized growth curve and its physiological interpretation by the introduction of proper age, which is proportional to the chronological age, are presented here. In the second phase, but not in the first phase, this constant of proportionality is highly correlated with the age at menarche.     

Glutathione Reductase-null Malaria Parasites Have Normal Blood Stage Growth but Arrest during Development in the Mosquito

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.     

Glutathione Content in Peach Buds in Relation to Development and Release of Rest

Glutathione content was determined in buds of one-year-old pottedpeach (Prunus persica L.) trees during rest development andrelease from rest. The content of reduced (GSH) and oxidizedglutathione (GSSG) changed with the accumulation of chillingunits. GSH and GSSG content decreased in the early phases ofrest, and then increased at maximum rest. GSH content continuedto increase and peaked on 1 Dec at 860 chill units, and thendropped during the quiescent stage. It appears that the increaseof GSH during chilling was closely associated with the breakingof rest. In contrast, GSSG showed only slight increase fromOct to Dec. Glutathione levels induced by the rest-breakingchemical, hydrogen cyanamide, were also studied throughout therest period. Five concentrations of cyanamide (0, 0.125, 0.25,0.5, and 1.0 M) were applied on 1 Oct, 15 Oct, 1 Nov, 15 Nov,1 Dec, and 15 Dec, 1990. Cyanamide treatments caused a depletionof GSH within 12 h followed by a large increase 24 h after treatment,whereas the untreated plants showed a relatively constant levelof GSH and GSSG during this time. The changes in GSH contentinduced by cyanamide were inversely related to the cyanamideconcentration applied. It appears that the extent of GSH changewas dependent on both the physiological status of the bud andthe cyanamide concentration. At maximum rest, the plants weremore resistant to cyanamide treatment and this coincided withthe highest level of cyanamide-induced GSH. ¹ Present address: Research Center in Food and Development,Apartado Postal 1735, Hermosillo, Sonora 83200, Mexico.     

Optimisation of Potassium Chloride Nutrition for Proper Growth,Physiological Development and Bioactive Component Production in Prunella vulgaris L

Prunella vulgaris L. is an important medicinal plant with a variety of pharmacological activities, but limited information is available about its response to potassium chloride (KCl) supplementation. P. vulgaris seedlings were cultured in media with four different KCl levels (0, 1.00, 6.00 and 40.00 mM). Characteristics relating to the growth, foliar potassium, water and chlorophyll content, photosynthesis, transpiration, nitrogen metabolism, bioactive constituent concentrations and yield were determined after three months. The appropriate KCl concentration was 6.00 mM to result in the highest values for dry weight, shoot height, spica and root weight, spica length and number in P. vulgaris. The optimum KCl concentration resulted in a maximum net photosynthetic rate (Pn) that could be associated with the highest chlorophyll content and fully open stomata conductance. A supply of surplus KCl resulted in a higher concentration of foliar potassium and negatively correlated with the biomass. Plants that were treated with the appropriate KCl level showed a greater capacity for nitrate assimilation. The Pn was significantly and positively correlated with nitrate reductase (NR) and glutamine synthetase (GS) activities and was positively correlated with leaf-soluble protein and free amino acid (FAA) contents. Both KCl starvation (0 mM) and high KCl (40.00 mM) led to water loss through a high transpiration rate and low water absorption, respectively, and resulted in increased concentrations of ursolic acid (UA), oleanolic acid (OA) and flavonoids, with the exception of rosmarinic acid (RA). Moreover, the optimum concentration of KCl significantly increased the yields of RA, UA, OA and flavonoids. Our findings suggested that significantly higher plant biomass; chlorophyll content; Pn; stronger nitrogen anabolism; lower RA, UA, OA and flavonoid accumulation; and greater RA, UA, OA and flavonoid yields in P. vulgaris could be expected in the presence of the appropriate KCl concentration (6.00 mM).     

Tissue Stiffness Dictates Development,Homeostasis, and Disease Progression

Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression.     

The Dehydratase ADT3 Affects ROS Homeostasis and Cotyledon Development

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling.During the transition from seed to seedling, the coordination of defense and development is critical for early survival (Finch-Savage and Leubner-Metzger, 2006; Holdsworth et al., 2008). After emerging from the seed coat, the embryo pushes through the soil to reach the surface; at this time, it is more vulnerable to biotic and abiotic stresses (Raven et al., 2005), and underlying actors of this transition are relatively unstudied (Warpeha and Montgomery, 2016). Phe-derived compounds, the phenylpropanoids, play an important role in the first line of defense by contributing to the reinforcement of the external cuticle layer and by conferring UV light protection properties to epicuticular waxes (Steyn et al., 2002; Pollard et al., 2008); in addition, phenylpropanoids influence wax production in response to UV light exposure (Rozema et al., 2002; Pollard et al., 2008; Warpeha et al., 2008). The activity of the phenylpropanoid pathway provides an additional line of defense, as phenolic compounds take part in a nonenzymatic mechanism to efficiently scavenge reactive oxygen species (ROS), whose levels increase as a result of metabolic reactions and when plants initiate a stress response (Sharma et al., 2012; Agati et al., 2013). Moreover, by influencing the cell’s ability to balance and modulate ROS production and scavenging, phenylpropanoids allow fluctuations in ROS levels that are required to elicit stress signaling pathways for specific defense strategies (Apel and Hirt, 2004; Mittler et al., 2011).AROGENATE DEHYDRATASE3 (ADT3)/PREPHENATE DEHYDRATASE1 belongs to the arogenate dehydratase protein family, whose members catalyze the last steps of the biosynthesis of Phe (Warpeha et al., 2006; Cho et al., 2007; Tzin and Galili, 2010; Bross et al., 2011). Activation of ADT3 leads to an increase in Phe content and in the production of phenylpropanoids (Warpeha et al., 2006). Accordingly, loss of ADT3 results in an enhanced sensitivity to UV irradiation in etiolated seedlings due to the reduced synthesis of photoprotective compounds and UV light-scattering epicuticular waxes (Warpeha et al., 2008). However, the physiological and molecular bases of this phenotype and the function of ADT3 in the seed-to-seedling transition remain to be elucidated.We sought to understand the role of ADT3 postgermination, in the seed-to-seedling transition. ADT3 is expressed early in seedling growth (Warpeha et al., 2006; Hruz et al., 2008). Localization studies in Arabidopsis (Arabidopsis thaliana) using protoplasts from cell suspension and light-grown rosette leaves have placed this enzyme within the chloroplast (Rippert et al., 2009), while we have reported ADT3 activity in the cytosolic fraction in young etiolated seedlings (Warpeha et al., 2006). These reports differ likely due to the different age and growth conditions of the studied plant material. Cytosolic forms of chorismate mutase, which act at the first committed step in the Phe and Tyr biosynthesis pathway, were found in Arabidopsis and other plants (d’Amato et al., 1984; Benesova and Bode, 1992; Eberhard et al., 1996), suggesting the possibility of extraplastidic Phe biosynthesis.Here, we report in transgenic complementation experiments that ADT3 is expressed widely in the young shoot and largely accumulates in the cytosol. Based on the role of phenylpropanoids in plant defense, we hypothesize that the ADT3 regulation of Phe supply is required to coordinate defense and development at the seed-to-seedling transition. We found that, without ADT3, the cells of the epidermis cannot buffer and restrict ROS; moreover, adt3 cotyledons enter an aberrant developmental program that results in abnormal morphology and patterning as well as several alterations at the subcellular level. Proteomic analysis of adt3 seedlings provided insights into the molecular basis of adt3 phenotypes, as it uncovered a chronic inability to buffer an excess of ROS and maintain plastid integrity. It also revealed a failed attempt to control cell rheology through up-regulation of the biochemical pathway for cuticle biosynthesis and assembly, as indicated by the increase in the permeability of adt3 epidermal cells; we propose that this also could be the cause of the defecting epidermal patterning in adt3 cotyledons. In addition, we suggest an additional role of Phe in nutrient supply in etiolated seedlings.     

Proteolysis and binding of myosin subfragment 1 to actin

Rates of proteolytic cleavage of myosin subfragment 1 were measured in the absence and presence of different amounts of actin. The rates of tryptic digestion at the 50K/20K junction and papain digestion at the 25K/50K junction of the myosin head were progressively inhibited with increasing substoichiometric molar ratios of actin to myosin subfragment 1. The percentage inhibitions of digestion reactions corresponded precisely to the molar compositions of actin-subfragment 1 solutions and demonstrated that equimolar complexes of these proteins were responsible for the observed changes in the proteolysis of myosin heads.     

Growth of Bradyrhizobium sp. SEMIA 6144 in Response to Methylglyoxal: Role of Glutathione

We previously showed the important role of glutathione (GSH) in the protection mechanism against different stresses, such as acid pH, saline, and oxidative stress, using a GSH-deficient mutant of Bradyrhizobium sp. (peanut microsymbiont). In this work, we studied the role of GSH in the protection mechanism against methylglyoxal (MG) toxicity. MG is a naturally occurring toxic electrophilic compound, and it has been shown that GSH is involved in the detoxification of MG in Escherichia coli. One recognized component of this detoxification process is the formation of a GSH adduct, which in turn transports potassium (K⁺) out of bacterial cells. Our results showed that growth of wild-type strain Bradyrhizobium sp. SEMIA 6144 was not affected at a MG concentration of 0.5 mM in the yeast extract–mannitol culture medium. However, a reduction of growth, at concentrations of 1.5 and 2.5 mM MG and reaching complete growth inhibition at 3.0 mM MG, was observed. In wild-type strain, intracellular GSH content decreased, and intracellular K⁺ content was unchanged, whereas GSH-deficient mutant SEMIA 6144-S7Z was unable to grow at 1.5 mM MG. The addition of external GSH to the incubation medium did not restore the growth rate either in wild-type or mutant strains. Our findings showed that GSH has not proven to be protective against the cell-growth inhibiting activity of MG. Therefore, the response of Bradyrhizobium sp. growth to MG is different from that reported in E. coli and other Gram-negative bacteria.     

Oncogenic IDH1 Mutations Promote Enhanced Proline Synthesis through PYCR1 to Support the Maintenance of Mitochondrial Redox Homeostasis

1. Download high-res image (183KB)
2. Download full-size image

     

CSF-1 Receptor-Dependent Colon Development,Homeostasis and Inflammatory Stress Response

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1^op/op mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r^−/− and Csf1^op/op mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r^−/− colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r ^+/− male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r ^+/− female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.