The impact of digestible lysine and sulfur amino acids on eggshell quality and egg weight control in old ISA brown hens during 62 to 74 wk.

Modifying dietary amino acids has been proposed as a strategy to improve eggshell quality by slowing down increases in egg weight (EW). This study aimed to investigate the effects of different levels of digestible lysine (dLYS) and ratios of digestible sulfur amino acids (dTSAA) to dLYS on performance and eggshell quality in ISA brown hens. A total of 288 hens were individually housed and assigned to 8 treatments, which combined 2 levels of dLYS (5.9 and 5.5 g/kg) with 4 ratios of dTSAA:dLYS (90, 85, 80, and 75) in a factorial arrangement. The study lasted 12 wk, starting at 62 wk of age. The number of eggs was not affected by the interaction between dLYS and dTSAA:dLYS or their main effect. However, the interaction between dLYS and dTSAA:dLYS showed that reducing the dTSAA:dLYS ratio from 85 to 75 when hens were fed 5.5 g/kg of dLYS resulted in a lower EW. Conversely, when hens were fed 5.9 g/kg of dLYS, no significant difference was found in EW among the different ratios of dTSAA:dLYS. Although there was no interaction between the levels of dLYS and dTSAA:dLYS on eggshell quality, reducing the dLYS level from 5.9 to 5.5 slowed down the deterioration in eggshell-breaking strength and eggshell thickness, regardless of the dTSAA:dLYS ratio. These findings suggest that adjusting dietary dLYS while maintaining the dTSAA:dLYS ratio of no less than 85 may be an effective strategy for decelerating the deterioration of eggshell quality in laying hen operations without impacting the egg production rate.

Effects of Lanthionine Ketimine-5-Ethyl Ester on the α-Synucleinopathy Mouse Model.

Potentially druggable mechanisms underlying synaptic deficits seen in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are under intense interrogations. In addition to defective synaptic vesicle trafficking, cytoskeletal disruption, autophagic perturbation, and neuroinflammation, hyperphosphorylation of microtubule-associated protein collapsin response mediator protein 2 (CRMP2, also known as DPYSL2) is newly determined to correlate with synaptic deficits in human DLB. The small molecule experimental therapeutic, lanthionine ketimine-5-ethyl ester (LKE), appears to interact with CRMP2 in a host of neurodegenerative mouse models, normalizing its phosphorylation level while promoting healthful autophagy in cell culture models and suppressing the proinflammatory phenotype of activated microglia. Accordingly, this study examined the effect of LKE on α-synuclein A53T transgenic (Tg) mice which were employed as a DLB model. We found that chronic administration of LKE to A53T mice suppressed (1) the accumulation of LBs, (2) neuroinflammatory activation of microglia, (3) impairment of contextual fear memory, and (4) CRMP2 phosphorylation at Thr509 in A53T Tg mice. These results suggest that CRMP2 phosphorylation by GSK3ß in the hippocampus is related to pathology and memory impairment in DLB, and LKE may have clinical implications in the treatment of α-synucleinopathy.

The effect of total sulfur amino acid levels on growth performance and bone metabolism in pullets under heat stress.

A study was conducted to investigate the effects of total sulfur amino acid (TSAA) levels on performance and bone metabolism in pullets under heat stress (HS). Hy-Line W36 day-old pullets (n = 216) were randomly distributed in 3 dietary treatments (70, 85, and 100% of TSAA), with 6 replicates of 12 birds. The treatments were defined as percentages of the TSAA level recommendation (100, 85, and 70%), and 85 and 100% of TSAA were obtained by adding L-Methionine to the basal deficient diet (70% of TSAA). The birds were raised under HS (35°C/7 h/D) from 1 to 18 wk. At 6, 12, and 18 wk, growth performance was measured. At 12 and 18 wk, bone weight, ash, collagenous (ColP), and noncollagenous proteins (NColP), tissue volume (TV), bone mineral content (BMC), and mineral density from total, cortical, and trabecular bones were evaluated. The means were subjected to ANOVA and, when significant (P ≤ 0.05), were compared by Dunnett's test. Regression analyses were performed to evaluate trends of TSAA dose response. Overall, birds fed 70% of TSAA showed poor growth and feed efficiency compared with other groups. Additionally, in at least 1 phase, birds fed 70% of TSAA showed lower bone ash, NColP, total BMC, and TV and higher ColP than the other treatments, whereas the cortical and trabecular TV and BMC were lower than 100% of TSAA (P < 0.04). Quadratic effects of TSAA levels on body weight gain (BWG) were found, and the level for maximum BWG was 95% of the TSAA recommendation (P < 0.03, R2 > 0.83). In conclusion, the use of a TSAA-deficient diet resulted in poor performance and delayed bone development. Additionally, the use of 100% of TSAA led to better initial structural bone development than 85% of TSAA. Therefore, the TSAA level recommended by the primary breeder guideline was enough to support growth and bone quality under HS, suggesting that HS does not alter TSAA requirement in pullets.

The effects of total sulfur amino acids on the intestinal health status of broilers challenged with Eimeria spp.

This study evaluated the effects of total sulfur amino acid (TSAA) levels on the performance and intestinal health of broilers challenged with Eimeria spp. A total of 432 one-day-old off-sex Cobb 500 male chicks were randomly assigned to a 3 × 2 factorial arrangement (6 replicates/12 birds), with diets and Eimeria challenge as the main factors. The diets were as follows: 70% (no methionine [Met] supplementation), 85, and 100% TSAA, supplemented with L-Met. At day 14, the challenged birds (n = 216) were orally gavaged with a pool of Eimeria acervulina, Eimeria maxima, and Eimeria tenella sporulated oocysts, and the unchallenged birds (n = 216) received water. At 6 and 12 D post inoculation (dpi), performance and intestinal health were evaluated. The challenge, regardless of diets, significantly impaired the performance, intestinal villi height, villus-to-crypt ratio, and ileal digestibility of dry matter, energy, and crude protein (CP) and modulated the tight junction protein (TJP) expression throughout the experiment. Moreover, the superoxide dismutase activity was increased, whereas the reduced glutathione (GSH)-to-oxidized glutathione (GSSG) ratio was decreased by the challenge at 6 dpi. Regardless of the challenge, the 70% TSAA diet reduced the body weight and feed intake in all phases, whereas the ileal digestibility of CP was higher in birds fed with the 70% TSAA diet than in those fed with the 100% TSAA diet at 6 dpi. No major differences were observed among the diets with regard to the intestinal histomorphology and TJP expression, and birds fed with the 100% TSAA diet had the highest GSH concentration at 12 dpi. Few interactions were observed, and the Met supplementation counteracted the negative effects of the Eimeria challenge on GSH concentration when 85 and 100% of TSAA levels were reached. Overall, the Eimeria challenge had a negative impact on growth and intestinal health. Moreover, the supplementation of L-Met until either 85 or 100% of TSAA levels were reached was enough to assure good performance and intestinal health in birds challenged or not challenged with Eimeria spp.

In vitro inhibition of Saccharomyces cerevisiae growth by Metschnikowia spp. triggered by fast removal of iron via two ways.

Simple and convenient innovative assays in vitro demonstrating Metschnikowia spp. competition with Saccharomyces cerevisiae for an essential nutrient iron are presented. The tested Metschnikowia strains possess a common genetically determined property of secreting a pulcherriminic acid which in the presence of iron (III) ions forms an insoluble red pigment pulcherrimin. Both initial accumulation in growing Metschnikowia cells and subsequent precipitation in the form of pulcherrimin in the media contribute to iron removal by functioning cells. The predominant way depends on the strain. Due to fast elimination of iron, the growth of S. cerevisiae can be inhibited by tested Metschnikowia strains at concentrations of elemental iron in the media not exceeding 12 mg kg-1. Inhibition can be regulated by additional supply of microquantities of iron onto the surface of the solid medium within 20-24 h. At relatively low concentrations of elemental iron (below 1 mg kg-1), additional supplements of iron onto the surface provide an advancement in understanding the inhibition possibilities and enable the assay control. Microscopy observations revealed that Metschnikowia chlamydospores are involved in iron removal at relatively high iron concentrations. The results may find application in development of new methodologies and strategies for biocontrol or inhibition of pathogenic microorganisms.

Cassane Diterpenoids from the Aerial Parts of Caesalpinia pulcherrima and Their Antifeedant and Insecticidal Activities against Mythimna separate and Plutella xylostella.

Ten new cassane diterpenoids, caesalpulcherrins A-J (1-10), together with 11 known analogues (11-21) were isolated from the aerial parts of Caesalpinia pulcherrima. Their structures and relative stereochemistry were elucidated by spectrometric and spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and single-crystal X-ray diffraction analysis. Compounds 1-4 represent the first examples of 2,5-dimethoxyfuranocassane diterpenoids. Results of the antifeedant activity indicated that isovouacapenol C (12) and pulcherrin N (14) exhibited remarkable antifeedant activity against Mythimna separate with EC50 values of 3.43 and 4.20 µg/cm2, respectively. Meanwhile, pulcherrimin C (13) and 12-demethyl neocaesalpin F (18) exerted significant antifeedant activity against Plutella xylostella with an EC50 data of 4.00 and 3.05 µg/cm2, respectively. Some of the compounds showed obvious toxic activity against the plant-feeding generalist insect herbivores, M. separate and P. xylostella, at 0.8 mg/mL (800 ppm). Furthermore, the structure-activity relationships of antifeedant and insecticidal activities are also discussed in the article.

Effects of the CRMP2 activator lanthionine ketimine ethyl ester on oligodendrocyte progenitor cells.

We previously showed LKE (lanthionine ketimine ester) reduces disease in the EAE model of multiple sclerosis, however whether LKE affects oligodendrocytes (OLGs) was not tested. In OLG progenitor cells (OPCs), LKE increased process number and area, but not PDGF-receptor-alpha expressing cells. In contrast, PDGF increased OPC numbers, but reduced process number and area. LKE increased collapsin response mediator protein-2 (CRMP2) expression, an LKE target, and CRMP2-expressing OLGs expressed myelin basic protein. LKE increased markers of OPC maturation, while PDGF, but not LKE, increased Sox2 expression. Our findings suggest that effects on OPCs may contribute to LKE beneficial actions in EAE.

Dietary sulfur amino acids affect jejunal cell proliferation and functions by affecting antioxidant capacity, Wnt/ß-catenin, and the mechanistic target of rapamycin signaling pathways in weaning piglets.

Intestinal epithelial cells undergo rapid renewal along the crypt-villus axis (CVA), which ensures intestinal functions. Weaning stress differentially effects intestinal epithelial cell metabolism and physiological states along the CVA. Sulfur amino acids (SAA) play a key role in intestinal epithelial cell functioning. This study evaluated the effects of SAA dietary supplementation on weaning pig jejunal epithelial cells along the CVA. Sixteen Duroc × Landrace × Yorkshire piglets (6.16 ± 0.22 kg BW) were weaned at 21 d of age and were blocked by BW and gender and the randomly assigned to 1 of 2 groups fed diets consisting of low (0.53%) or high (0.85%) levels of SAA for a 7-d period. All piglets were euthanized for tissue sampling on day 7 postweaning. Jejunal epithelial cells were isolated along the CVA to yield 3 "cell fractions" (upper villus, middle villus, and crypt cells). The number of proliferating cells per crypt of piglets fed the high SAA diet was lower (P < 0.05) than that for low SAA diet. High SAA diet piglets tended to have decreased (P = 0.059) sucrase activities compared low SAA diet piglets. A high SAA diet increased (P < 0.05) total antioxidant capacity, catalase, and superoxide dismutase activities compared with a low SAA diet. mRNA expression levels of claudin-1, Slc5a1, and Slc7a9 in high SAA diet piglets were lower (P < 0.05) than for low SAA diet piglets. There were no interactions between dietary SAA and cell sections along the CVA for enzyme activities and mRNA expression in any of the weaned piglets. Protein amounts and phosphorylation levels related to Wnt/ß-catenin and mechanistic targeting of rapamycin (mTOR) signaling pathways were affected by SAA in weaning piglets. These findings indicate that dietary SAA affects jejunal cell proliferation and functions in weaning piglets. There appears to be no interactions between dietary SAA and cell sections along the CVA. The effects of SAA may be partly through affecting antioxidant capacity, and Wnt/ß-catenin and mTOR signaling pathway.

Lanthionine ketimine-5-ethyl ester provides neuroprotection in a zebrafish model of okadaic acid-induced Alzheimer's disease.

Okadaic acid (OKA) is a protein phosphatase 2A inhibitor that is used to induce neurodegeneration and study disease states such as Alzheimer's disease (AD). Lanthionine ketimine-5-ethyl ester (LKE) is a bioavailable derivative of the naturally occurring brain sulfur metabolite, lanthionine ketimine (LK). In previously conducted studies, LKE exhibited neuroprotective and neurotrophic properties in murine models but its mechanism of action remains to be clarified. In this study, a recently established zebrafish OKA-induced AD model was utilized to further elucidate the neuroprotective and neurotrophic properties of LKE in the context of an AD-like condition. The fish were divided into 3 groups containing 8 fish per group. Group #1 = negative control, Group #2 = 100 nM OKA, Group #3 = 100 nM OKA +500 µM LKE. OKA caused severe cognitive impairments in the zebrafish, but concomitant treatment with LKE protected against cognitive impairments. Further, LKE significantly and substantially reduced the number of apoptotic brain cells, increased brain-derived neurotrophic factor (BDNF), and increased phospho-activation of the pro-survival factors pAkt (Ser 473) and pCREB (Ser133). These findings clarify the neuroprotective and neurotrophic effects of LKE by highlighting particular survival pathways that are bolstered by the experimental therapeutic LKE.

Multiple-step, one-pot synthesis of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates and their corresponding ethyl esters.

The multiple-step, one-pot procedure for a series of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates, analogues of the natural, sulfur amino acid metabolite lanthionine ketimine (LK), its 5-ethyl ester (LKE) and 2-substituted LKEs is described. Initiating the synthesis with the Michaelis-Arbuzov preparation of α-ketophosphonates allows for a wide range of functional variation at the 2-position of the products. Nine new compounds were synthesized with overall yields range from 40 to 62%. In addition, the newly prepared 2-isopropyl-LK-P, 2-n-hexyl-LKE-P and 2-ethyl-LKE were shown to stimulate autophagy in cultured cells better than that of the parent compound, LKE.

The effects of dietary sulfur amino acids on growth performance, intestinal morphology, enzyme activity, and nutrient transporters in weaning piglets.

Early weaning results in intestinal dysfunction in piglets, while sulfur amino acids (SAA) are involved in improving intestinal functions. We tested a hypothesis that dietary supplementation with SAA can improve intestinal functions of weaning piglets and analyzed the effects of different dietary SAA levels on intestinal functions. A total of 80 piglets (Duroc × Landrace × Yorkshire) were weaned at 21 d of age and randomly assigned to one of the five diets that contained 0.53%, 0.63%, 0.74%, 0.85%, or 0.96% SAA, which corresponded to 70%, 85%, 100%, 115%, or 130% of the SAA:Lys ratio recommended by the National Research Council (2012). The 14 d feeding experiment involved 16 pens per diet and one piglet per pen. Eight randomly selected piglets from each treatment were euthanized for tissue sampling on day 7 and 14 post weaning. Supplementation with SAA led to a rise over time in G:F (linear, P = 0.001; quadratic, P = 0.001). Between day 0 and 14 of treatment, the jejunal crypt depth decreased (linear, P = 0.018; quadratic, P = 0.015), while that of the duodenal villus (linear, P = 0.049) and ileal villus width (linear, P = 0.029; quadratic, P = 0.034) increased. The activities of jejunal alkaline phosphatase (ALP) were quadratically increased (P = 0.040) from day 0 to 14 due to dietary SAA. Dietary SAA also elevated the activities of jejunal lactase (linear, P = 0.003; quadratic, P = 0.004), jejunal sucrase (linear, P = 0.032; quadratic, P = 0.027), and jejunal contents of glutathione (GSH) from day 0 to 7, as well as the activity of jejunal maltase (linear, P = 0.014; quadratic, P = 0.001) between day 0 and 14. During the first wk, dietary SAA linearly increased the amounts of intestinal-type fatty acid-binding protein (I-FABP) (P = 0.048) and SGLT-1 (P = 0.021) and linearly decreased the amount of GLUT2 (P = 0.029) proteins in the jejunum. The abundance of jejunal I-FABP (P = 0.044) and PEPT1 (P = 0.049) protein linearly increased from day 0 to 14 in response to this supplementation. These findings indicate that there is a dose-dependent response to dietary SAA on feed efficiency and intestinal parameters of weanling pigs.

Sulfur amino acid restriction-induced changes in redox-sensitive proteins are associated with slow protein synthesis rates.

The mechanisms underlying life span extension by sulfur amino acid restriction (SAAR) are unclear. Cysteine and methionine are essential for the biosynthesis of proteins and glutathione (GSH), a major redox buffer in the endoplasmic reticulum (ER). We hypothesized that SAAR alters protein synthesis by modulating the redox milieu. Male F344-rats were fed control (CD: 0.86% methionine without cysteine) and SAAR diets (0.17% methionine without cysteine) for 12 weeks. Growth rates, food intake, cysteine and GSH levels, proteins associated with redox status and translation, and fractional protein synthesis rates (FSRs) were determined in liver. Despite a 40% higher food intake, growth rates for SAAR rats were 27% of those fed CD. Hepatic free cysteine in SAAR rats was 55% compared with CD rats. SAAR altered tissue distribution of GSH, as hepatic and erythrocytic levels were 56% and 196% of those in CD rats. Lower GSH levels did not induce ER stress (i.e., unchanged expression of Xbp1s , Chop, and Grp78), but activated PERK and its substrates eIF2-α and NRF2. SAAR-induced changes in translation-initiation machinery (higher p-eIF2-α and 4E-BP1, and lower eIF4G-1) resulted in slower protein synthesis rates (53% of CD). Proteins involved in the antioxidant response (NRF2, KEAP1, GCLM, and NQO1) and protein folding (PDI and ERO1-α) were increased in SAAR. Lower FSR and efficient protein folding might be improving proteostasis in SAAR.

Neuroprotective and neurotrophic effects of Lanthionine Ketimine Ester.

Lanthionine ketimine ethyl ester (LKE) is a synthetic derivative of the naturally occurring amino acid lanthionine ketimine. We previously showed that LKE reduced clinical signs in a mouse model of multiple sclerosis (MS) associated with reductions in axonal damage; however, whether LKE has direct beneficial actions on mammalian neuronal cells was not examined. In the current study, we tested the effects of LKE in SH-SY5Y human neuronal cells and in primary mouse cerebellar granule neurons. In both cell types, LKE dose-dependently reduced the cell death that occurred spontaneously followed a change in media. LKE also reduced cell death due to glutamate excitoxicity, accompanied by a reduction in production of reactive oxygen species. LKE induced neuritogenesis in both undifferentiated SH-SY5Y cells and in primary neuron, increasing process numbers and lengths. These results demonstrate that direct neuroprotective and neurotrophic effects of LKE likely contribute to its beneficial actions in vivo.

Lanthionine ketimine ester promotes locomotor recovery after spinal cord injury by reducing neuroinflammation and promoting axon growth.

The mammalian central nervous system (CNS) has limited regenerative ability after injury, largely due to scar formation and axonal growth inhibitors. Experimental suppression of neuroinflammation encourages recovery from spinal cord injury (SCI), yet practical means for pharmacologically treating SCI have remained elusive. Lanthionine ketimine (LK) is a natural brain sulfur amino acid metabolite with demonstrated anti-neuroinflammatory and neurotrophic activities. LK and its synthetic brain-penetrating ethyl ester (LKE) promote growth factor-dependent neurite extension in cultured cell and suppress microglial activation in animal models of neurodegeneration. Thus far however, LKE has not been explored as a potential therapy for SCI. The present study investigated the hypothesis that systemic LKE could improve motor functional recovery after SCI in a mouse model. Intraperitoneal administration of LKE (100 mg/kg/d) after near-complete transect of spinal cord at the T7 level significantly improved motor function over a 4-week time course. Vehicle-treated mice, in contrast, demonstrated negligible functional recovery. In terms of histology, LKE treatment reduced pro-neuroinflammatory microglia/macrophage activation evidenced by quantitative Iba1 labeling and shifted the microglial phenotype toward a more neurotrophic M2 character evidenced by changes in the M2 marker arginase-1. This was correlated with less dense scar formation and more extensive axonal regrowth across the transection site demonstrated by 5-hydroxytryptamine (5HT) immunolabeling of raphespinal tract axons. These data provide evidence that LKE or similar compounds have potential therapeutic value for recovery after certain forms of SCI.

Autophagy Modulation by Lanthionine Ketimine Ethyl Ester Improves Long-Term Outcome after Central Fluid Percussion Injury in the Mouse.

Diffuse axonal injury is recognized as a progressive and long-term consequence of traumatic brain injury. Axonal injury can have sustained negative consequences on neuronal functions such as anterograde and retrograde transport and cellular processes such as autophagy that depend on cytoarchitecture and axon integrity. These changes can lead to somatic atrophy and an inability to repair and promote plasticity. Obstruction of the autophagic process has been noted after brain injury, and rapamycin, a drug used to stimulate autophagy, has demonstrated positive effects in brain injury models. The optimization of drugs to promote beneficial autophagy without negative side effects could be used to attenuate traumatic brain injury and promote improved outcome. Lanthionine ketimine ethyl ester, a bioavailable derivative of a natural sulfur amino acid metabolite, has demonstrated effects on autophagy both in vitro and in vivo. Thirty minutes after a moderate central fluid percussion injury and throughout the survival period, lanthionine ketimine ethyl ester was administered, and mice were subsequently evaluated for learning and memory impairments and biochemical and histological changes over a 5-week period. Lanthionine ketimine ethyl ester, which we have shown previously to modulate autophagy markers and alleviate pathology and slow cognitive decline in the 3 × TgAD mouse model, spared cognition and pathology after central fluid percussion injury through a mechanism involving autophagy modulation.

Effects of different sulphur amino acids and dietary electrolyte balance levels on performance, jejunal morphology, and immunocompetence of broiler chicks.

As alterations of dietary electrolyte balance (DEB) can influence amino acid metabolism via changes the ions incur in their configurations, performance and immunological responses of broiler chicks might be affected. So, the current study was carried out to investigate the effects of different levels of sulphur amino acids (SAA) and DEB on performance, jejunal morphology and immunocompetence of broiler chicks. A total of 360 1-day-old male Ross 308 broiler chicks were randomly assigned to nine experimental treatments with four replicates of 10 birds each. Experimental treatments consisted of three levels of SAA (100, 110, and 120% of NRC recommendation, provided by methionine supplementation in diets with the same cysteine level) and three levels of DEB (150, 250, and 350 mEq/kg) that were fed during the entire of trial in a 3 × 3 factorial arrangement. Results showed that the relative weights of intestine and abdominal fat were decreased markedly (p < 0.001) with increasing levels of SAA and DEB respectively. Antibody titre against sheep red blood cell was neither individually nor in combination influenced by supplementation of SAA or DEB. Nevertheless, a decrease in DEB level led to a suppression in heterophile (p < 0.05) and an increase in lymphocyte counts (p = 0.06); consequently, heterophile to lymphocyte ratio was significantly decreased (p < 0.05) by decremental levels of DEB. Albumin to globulin ratio was increased after inclusion of at least 10% SAA (p < 0.001) and 150 mEq DEB/kg in the diet (p = 0.11). Although feeding high-DEB level led to a remarkable decrease in villus height (p < 0.01) and goblet cell numbers (p < 0.001), supplementing the highest level of SAA improved the height of jejunal villus. During the entire trial period, average daily feed intake (ADFI) was increased by incremental SAA levels (p < 0.05). However, inclusion of 150 mEq/kg led to not only a remarkable increase (p < 0.0001) in both ADFI and average daily weight gain (ADWG) but also to improved (p < 0.001) feed conversion ratio (FCR) both during the growing and over the entire trial periods. The present findings indicated that inclusion of low DEB decreased the heterophile to lymphocyte ratio and improved both the albumin to globulin ratio and intestinal health indices. The best growth performance was obtained with 150 mEq DEB/kg in the diet for each level of SAA.

Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

Lanthionine ketimine ester provides benefit in a mouse model of multiple sclerosis.

Lanthionine ketimine (LK) is a natural sulfur amino acid metabolite which binds to collapsin response mediator protein-2 (CRMP2), an abundant brain protein that interacts with multiple partners to regulate microtubule dynamics, neurite growth and retraction, axonal transport, and neurotransmitter release. LK ethyl-ester (LKE) is a cell-permeable synthetic derivative that promotes neurogenesis, suppresses nitric oxide production from microglia, and reduces neurotoxicity of microglia-conditioned medium. These properties led us to test the effects of LKE in experimental autoimmune encephalomyelitis (EAE), a commonly used mouse model of multiple sclerosis. Female C57Bl/6 mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to develop a chronic disease. LKE was provided in the chow at 100 ppm, ad libitum beginning when the mice reached moderate clinical signs. Over the following 4 weeks the LKE-treated mice showed a significant reduction in clinical signs compared to vehicle-treated mice. LKE dose dependently reduced IFNγ production from splenic T cells, but had no effect on IL-17 production suggesting protective effects were mediated within the CNS. Electron microscopy revealed that, compared to sham mice, EAE mice had significant neurodegeneration in both the optic nerve and spinal cord, which was reduced in the LKE-treated mice. In contrast only minimal disruption of myelin was observed at this time point. In the optic nerve, measurements of axon caliber and myelin thickness showed little changes between sham and EAE mice, however, treatment with LKE increased the percentage of axons with thicker myelin and with larger axon calibers. In the spinal cord, only smaller effects of LKE on myelin thickness were observed. The effects of LKE were associated with a reduced relative level of phosphorylated CRMP2 to CRMP2. Together, these results demonstrate that LKE reduces neurodegeneration in a chronic EAE model of MS, which could have translation potential for treatment of progressive forms of MS.

A cell-penetrating ester of the neural metabolite lanthionine ketimine stimulates autophagy through the mTORC1 pathway: Evidence for a mechanism of action with pharmacological implications for neurodegenerative pathologies.

Autophagy is a fundamental cellular recycling process vulnerable to compromise in neurodegeneration. We now report that a cell-penetrating neurotrophic and neuroprotective derivative of the central nervous system (CNS) metabolite, lanthionine ketimine (LK), stimulates autophagy in RG2 glioma and SH-SY5Y neuroblastoma cells at concentrations within or below pharmacological levels reported in previous mouse studies. Autophagy stimulation was evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3) both in the absence and presence of bafilomycin-A1 which discriminates between effects on autophagic flux versus blockage of autophagy clearance. LKE treatment caused changes in protein level or phosphorylation state of multiple autophagy pathway proteins including mTOR; p70S6 kinase; unc-51-like-kinase-1 (ULK1); beclin-1 and LC3 in a manner essentially identical to effects observed after rapamycin treatment. The LKE site of action was near mTOR because neither LKE nor the mTOR inhibitor rapamycin affected tuberous sclerosis complex (TSC) phosphorylation status upstream from mTOR. Confocal immunofluorescence imaging revealed that LKE specifically decreased mTOR (but not TSC2) colocalization with LAMP2(+) lysosomes in RG2 cells, a necessary event for mTORC1-mediated autophagy suppression, whereas rapamycin had no effect. Suppression of the LK-binding adaptor protein CRMP2 (collapsin response mediator protein-2) by means of shRNA resulted in diminished autophagy flux, suggesting that the LKE action on mTOR localization may occur through a novel mechanism involving CRMP2-mediated intracellular trafficking. These findings clarify the mechanism-of-action for LKE in preclinical models of CNS disease, while suggesting possible roles for natural lanthionine metabolites in regulating CNS autophagy.