Matrix metalloproteinases (MMPs) are critical for development, wound healing, and for the progression of cancer. It is generally accepted that MMPs are secreted in a latent form (proMMP) and are activated only upon removal of their inhibitory propeptides. This report shows that three members of the SIBLING (Small, Integrin-Binding LIgand, N-linked Glycoprotein) family can specifically bind (Kd approximately equal nM) and activate three different MMPs. Binding of SIBLING to their corresponding proMMPs is associated with structural changes as indicated by quenching of intrinsic tryptophan fluorescence, increased susceptibility to plasmin cleavage, and decreased inhibition by specific natural and synthetic inhibitors. Activation includes both making the proMMPs enzymatically active and the reactivation of the TIMP (tissue inhibitors of MMP) inhibited MMPs. Bone sialoprotein specifically binds proMMP-2 and active MMP-2, while osteopontin binds proMMP-3 and active MMP-3, and dentin matrix protein-1 binds proMMP-9 and active MMP-9. Both pro and active MMP-SIBLING complexes are disrupted by the abundant serum protein, complement Factor H, thereby probably limiting SIBLING-mediated activation to regions immediately adjacent to sites of secretion in vivo. These data suggest that the SIBLING family offers an alternative method of controlling the activity of at least three MMPs.

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of beta-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent beta-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc(Min/+) mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP-(cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.

The importance of the gut-brain axis in regulating stress-related responses has long been appreciated. More recently, the microbiota has emerged as a key player in the control of this axis, especially during conditions of stress provoked by real or perceived homeostatic challenge. Diet is one of the most important modifying factors of the microbiota-gut-brain axis. The routes of communication between the microbiota and brain are slowly being unravelled, and include the vagus nerve, gut hormone signaling, the immune system, tryptophan metabolism, and microbial metabolites such as short chain fatty acids. The importance of the early life gut microbiota in shaping later health outcomes also is emerging. Results from preclinical studies indicate that alterations of the early microbial composition by way of antibiotic exposure, lack of breastfeeding, birth by Caesarean section, infection, stress exposure, and other environmental influences - coupled with the influence of host genetics - can result in long-term modulation of stress-related physiology and behaviour. The gut microbiota has been implicated in a variety of stress-related conditions including anxiety, depression and irritable bowel syndrome, although this is largely based on animal studies or correlative analysis in patient populations. Additional research in humans is sorely needed to reveal the relative impact and causal contribution of the microbiome to stress-related disorders. In this regard, the concept of psychobiotics is being developed and refined to encompass methods of targeting the microbiota in order to positively impact mental health outcomes. At the 2016 Neurobiology of Stress Workshop in Newport Beach, CA, a group of experts presented the symposium "The Microbiome: Development, Stress, and Disease". This report summarizes and builds upon some of the key concepts in that symposium within the context of how microbiota might influence the neurobiology of stress.

Glutaryl-CoA dehydrogenase (GCDH) deficiency is a rare inborn disorder of L-lysine, L-hydroxylysine, and L-tryptophan metabolism complicated by striatal damage during acute encephalopathic crises. Three decades after its description, the natural history and how to treat this disorder are still incompletely understood. To study which variables influenced the outcome, we conducted an international cross-sectional study in 35 metabolic centers. Our main outcome measures were onset and neurologic sequelae of acute encephalopathic crises. A total of 279 patients (160 male, 119 female) were included who were diagnosed clinically after clinical presentation (n = 218) or presymptomatically by neonatal screening (n = 23), high-risk screening (n = 24), or macrocephaly (n = 14). Most symptomatic patients (n = 185) had encephalopathic crises, characteristically resulting in bilateral striatal damage and dystonia, secondary complications, and reduced life expectancy. First crises usually occurred during infancy (95% by age 2 y); the oldest age at which a repeat crisis was reported was 70 mo. In a few patients, neurologic disease developed without a reported crisis. Differences in the diagnostic criteria and therapeutic protocols for patients with GCDH deficiency resulted in a huge variability in the outcome worldwide. Recursive partitioning demonstrated that timely diagnosis in neurologically asymptomatic patients followed by treatment with L-carnitine and a lysine-restricted diet was the best predictor of good outcome, whereas treatment efficacy was low in patients diagnosed after the onset of neurologic disease. Notably, the biochemical phenotype did not predict the clinical phenotype. Our study proves GCDH deficiency to be a treatable disorder and a good candidate for neonatal screening.

The kynurenine pathway of tryptophan metabolism accounts for most of the tryptophan that is not committed to protein synthesis and includes compounds active in the nervous and immune systems. Kynurenine acts on the aryl hydrocarbon receptor, affecting the metabolism of xenobiotics and promoting carcinogenesis. Quinolinic acid is an agonist at N-methyl-D-aspartate receptors (NMDARs), but is also pro-oxidant, has immunomodulatory actions, and promotes the formation of hyperphosphorylated tau proteins. Kynurenic acid blocks NMDARs and α7-homomeric nicotinic cholinoceptors and is also an agonist at the orphan G-protein-coupled receptor GPR35. 3-Hydroxykynurenine and 3-hydroxyanthranilic acid have pronounced redox activity and regulate T cell function. Cinnabarinic acid can activate metabotropic glutamate receptors. This review highlights the increasing range of molecular targets for components of the kynurenine pathway in both the nervous and immune systems in relation to their relevance to disease and drug development.

Elicitors from the plant pathogen Erwinia carotovora trigger coordinate induction of the tryptophan (Trp) biosynthesis pathway and Trp oxidizing genes in Arabidopsis. To elucidate the biological role of such pathogen-induced activation we characterized the production of secondary defense metabolites such as camalexin and indole glucosinolates derived from precursors of this pathway. Elicitor induction was followed by a specific increase in 3-indolylmethylglucosinolate (IGS) content, but only a barely detectable accumulation of the indole-derived phytoalexin camalexin. The response is mediated by jasmonic acid as shown by lack of IGS induction in the jasmonate-insensitive mutant coi1-1. In accordance with this, methyl jasmonate was able to trigger IGS accumulation in Arabidopsis. In contrast, ethylene and salicylic acid seem to play a minor role in the response. They did not trigger alterations in IGS levels, and methyl jasmonate- or elicitor-induced IGS accumulation in NahG and ethylene-insensitive ein2-1 mutant plants was similar as in the wild type. The breakdown products of IGS and other glucosinolates were able to inhibit growth of E. carotovora. The results suggest that IGS is of importance in the defense against bacterial pathogens.

BACKGROUND

Human ageing is a complex, multifactorial process and early developmental factors affect health outcomes in old age.

METHODS

Metabolomic profiling on fasting blood was carried out in 6055 individuals from the UK. Stepwise regression was performed to identify a panel of independent metabolites which could be used as a surrogate for age. We also investigated the association with birthweight overall and within identical discordant twins and with genome-wide methylation levels.

RESULTS

We identified a panel of 22 metabolites which combined are strongly correlated with age (R(2) = 59%) and with age-related clinical traits independently of age. One particular metabolite, C-glycosyl tryptophan (C-glyTrp), correlated strongly with age (beta = 0.03, SE = 0.001, P = 7.0 × 10(-157)) and lung function (FEV1 beta = -0.04, SE = 0.008, P = 1.8 × 10(-8) adjusted for age and confounders) and was replicated in an independent population (n = 887). C-glyTrp was also associated with bone mineral density (beta = -0.01, SE = 0.002, P = 1.9 × 10(-6)) and birthweight (beta = -0.06, SE = 0.01, P = 2.5 × 10(-9)). The difference in C-glyTrp levels explained 9.4% of the variance in the difference in birthweight between monozygotic twins. An epigenome-wide association study in 172 individuals identified three CpG-sites, associated with levels of C-glyTrp (P < 2 × 10(-6)). We replicated one CpG site in the promoter of the WDR85 gene in an independent sample of 350 individuals (beta = -0.20, SE = 0.04, P = 2.9 × 10(-8)). WDR85 is a regulator of translation elongation factor 2, essential for protein synthesis in eukaryotes.

CONCLUSIONS

Our data illustrate how metabolomic profiling linked with epigenetic studies can identify some key molecular mechanisms potentially determined in early development that produce long-term physiological changes influencing human health and ageing.

Comorbid depression and chronic pain are highly prevalent in individuals suffering from physical illness. Here, we critically examine the possibility that inflammation is the common mediator of this comorbidity, and we explore the implications of this hypothesis. Inflammation signals the brain to induce sickness responses that include increased pain and negative affect. This is a typical and adaptive response to acute inflammation. However, chronic inflammation induces a transition from these typical sickness behaviors into depression and chronic pain. Several mechanisms can account for the high comorbidity of pain and depression that stem from the precipitating inflammation in physically ill patients. These mechanisms include direct effects of cytokines on the neuronal environment or indirect effects via downregulation of G protein-coupled receptor kinase 2, activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase that generates neurotropic kynurenine metabolites, increased brain extracellular glutamate, and the switch of GABAergic neurotransmission from inhibition to excitation. Despite the existence of many neuroimmune candidate mechanisms for the co-occurrence of depression and chronic pain, little work has been devoted so far to critically assess their mediating role in these comorbid symptoms. Understanding neuroimmune mechanisms that underlie depression and pain comorbidity may yield effective pharmaceutical targets that can treat both conditions simultaneously beyond traditional antidepressants and analgesics.

The kynurenine pathway (KP) metabolizes the essential amino acid tryptophan and generates a number of neuroactive metabolites collectively called the kynurenines. Segregated into at least two distinct branches, often termed the "neurotoxic" and "neuroprotective" arms of the KP, they are regulated by the two enzymes kynurenine 3-monooxygenase and kynurenine aminotransferase, respectively. Interestingly, several enzymes in the pathway are under tight control of inflammatory mediators. Recent years have seen a tremendous increase in our understanding of neuroinflammation in CNS disease. This review will focus on the regulation of the KP by inflammatory mediators as it pertains to neurodegenerative and psychiatric disorders.

Many insects rely on bacterial symbionts with tiny genomes specialized for provisioning nutrients lacking in host diets. Xylem sap and phloem sap are both deficient as insect diets, but differ dramatically in nutrient content, potentially affecting symbiont genome evolution. For sap-feeding insects, sequenced symbiont genomes are available only for phloem-feeding examples from the suborder Sternorrhyncha and xylem-feeding examples from the suborder Auchenorrhyncha, confounding comparisons. We sequenced genomes of the obligate symbionts, Sulcia muelleri and Nasuia deltocephalinicola, of the phloem-feeding pest insect, Macrosteles quadrilineatus (Auchenorrhyncha: Cicadellidae). Our results reveal that Nasuia-ALF has the smallest bacterial genome yet sequenced (112 kb), and that the Sulcia-ALF genome (190 kb) is smaller than that of Sulcia in other insect lineages. Together, these symbionts retain the capability to synthesize the 10 essential amino acids, as observed for several symbiont pairs from xylem-feeding Auchenorrhyncha. Nasuia retains genes enabling synthesis of two amino acids, DNA replication, transcription, and translation. Both symbionts have lost genes underlying ATP synthesis through oxidative phosphorylation, possibly as a consequence of the enriched sugar content of phloem. Shared genomic features, including reassignment of the UGA codon from Stop to tryptophan, and phylogenetic results suggest that Nasuia-ALF is most closely related to Zinderia, the betaproteobacterial symbiont of spittlebugs. Thus, Nasuia/Zinderia and Sulcia likely represent ancient associates that have co-resided in hosts since the divergence of leafhoppers and spittlebugs >200 Ma, and possibly since the origin of the Auchenorrhyncha, >260 Ma.

The membrane-proximal tryptophan-rich region of the HIV transmembrane glycoprotein, gp41, plays an important role in the membrane fusion reaction. Using NMR spectroscopy, we have studied the tertiary structure of a synthetic 19-residue amidated peptide (NH2-KWASLWNWFNITNWLWYIK-CONH2) corresponding to this region in membrane-mimetic environments. Initial experiments in sodium dodecyl sulfate/H2O micelles and trifluoroethanol gave poor results, because of low solubility. However, in dodecylphosphocholine micelles, we obtained excellent 500 and 800 MHz NMR spectra, suggesting that the peptide has a preference for a zwitterionic membrane-like environment. The final NMR structures demonstrated a well-defined helical peptide with a backbone rmsd of 0.47 +/- 0.18 A. Four of the five tryptophan residues, as well as the tyrosine residue, formed a "collar" of aromatic residues along the axial length of the helix. By analogy to related tryptophan-rich antimicrobial peptides, the structure indicates that the aromatic residues of the HIV peptide are positioned within the membrane-water interface of a phospholipid bilayer. This is confirmed by the observation of direct NOEs between the aromatic residues of the peptide to the headgroup and interfacial protons of prototonated dodecylphosphocholine. The bulk of the polar residues are positioned on one face of this structure, with the hydrophobic phenylalanine side chain on the opposing face, forming an amphipathic structure. This work shows that the Trp-rich membrane-proximal region of HIV and related viruses can bind to the surfaces of zwitterioninc membranes in a "Velcro-like" manner.

Eukaryotic translation initiation factor eIF-4E plays a central role in the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthesis. eIF-4E exists in both phosphorylated and nonphosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eIF-4E phosphorylation facilitates its participation in protein synthesis. However, the biochemical basis for the functional difference between the two forms of eIF-4E is unknown. To address this directly, we have developed a method for the separation of phosphorylated and nonphosphorylated eIF-4E from rabbit reticulocytes by chromatography on rRNA-Sepharose. Using the resultant purified forms, we have studied the protein's interaction with the cap analogs m7GTP and m7GpppG and with the cap of globin mRNA by fluorescence quenching of tryptophan residues. It was found that phosphorylated eIF-4E had 3- to 4-fold greater affinity for cap analogs and mRNA than nonphosphorylated eIF-4E. The equilibrium binding constants (x 10(5), expressed as M-1) for the interaction of phosphorylated eIF-4E with m7GTP, m7GpppG, and globin mRNA were 20.0 +/- 0.1, 16.4 +/- 0.1, and 31.0 +/- 0.1, respectively, whereas those for the nonphosphorylated form were 5.5 +/- 0.4, 4.3 +/- 0.4, and 10.0 +/- 0.1, respectively. Treatment with potato acid phosphatase converted the phosphorylated form to the nonphosphorylated form and decreased the binding constant for m7GTP by a factor of 3. The increased affinity for mRNA caps may account for the in vivo and in vitro correlations between eIF-4E phosphorylation and accelerated protein synthesis and cell growth.

Human platelet factor 4, a protein that binds heparin, has been purified to apparent homogeneity and the complete amino acid sequence of the protein has been determined. The 70-residue polypeptide chain contains no methionine, tryptophan, or phenylalanine, and contains only a single tyrosyl residue. The sequence analysis demonstrates a highly negatively charged amino-terminal region. The carboxyl-terminal region of the polypeptide is unusual in that it contains a repetitive clustering of positively charged and hydrophobic pairs of amino acids; preliminary evidence suggests that this domain may play a role in the binding of heparin.

A channel-forming antimicrobial peptide, magainin 2, has been shown to translocate across phospholipid bilayers by forming a pore comprising multimeric peptides. The translocation was demonstrated by four sets of experiments by use of resonance energy transfer from tryptophan introduced into the peptide to a dansyl chromophore incorporated into the lipid membrane. The translocation was coupled to pore formation, as detected by the dye efflux from the lipid vesicles; about 30% of the total peptide molecules translocated into the inner leaflets over 10 min, while 80% of the dye molecules leaked out at a lipid to peptide ratio of 57. This novel model can explain the problems debated so far, i.e., the peptide forms an ion channel whereas the magainin helix essentially lies parallel to the membrane surface. Channel (pore) formation in the vesicles is a transient process observable mainly during the early stage of the peptide membrane interactions.

Purified proteoglycans extracted from pig laryngeal cartilage in 0.15 M-NaCl and 4 M-guanidinium chloride were analysed and their amino acid compositions determined. Selective modification of amino acid residues on the protein core confirmed that binding to hyaluronate was a function of the protein core, and was dependent on disulphide bridges, intact arginine and tryptophan residues, and epsilon-amino groups of lysine. Fluorescence measurement suggested that tryptophan was not involved in direct subsite interactions with the hyaluronate. The polydispersity in size and heterogeneity in composition of the aggregating proteoglycan was compatible with a structure based on a protein core containing a globular hyaluronate-binding region and an extended region of variable length also containing a variable degree of substitution with chondroitin sulphate chains. The non-aggregated proteoglycan extracted preferentially in 0.15 M-NaCl, which was unable to bind to hyaluronate, contained less cysteine and tryptophan than did other aggregating proteoglycans and may be deficient in the hyaluronate-binding region. Its small average size and low protein and keratan sulphate contents suggest that it may be a fragment of the chondroitin sulphate-bearing region of aggregating proteoglycan produced by proteolytic cleavage of newly synthesized molecules before their secretion from the cell.

A mitochondrial mutation that genetically maps in the middle of the gene coding cytochrome c oxidase subunit II has been found to be a single-base-pair deletion. Three independently isolated spontaneous revertants of this mutant have different single-base-pair insertions within 15 nucleotides of the mutation. These findings clearly identify the location of the gene and suggest that the mutation causes a frame-shift. The sequence of about 900 base pairs surrounding the mutation has been determined and found to have several chain termination codons in every possible reading frame. The sequence can, however, be translated in one frame by assuming that the codon TGA does not cause chain termination in yeast mitochondira, as was recently suggested for the human organelle [Barrell, B. G., Bankier, A. T. & Drouin, J. (1979) Nature (London), in press]. If TGA codes for tryptophan residues, as is apparently the case in human mitochondria, a polypeptide can be read from the yeast mtDNA that is identical to bovine cytochrome oxidase subunit II at 37.8% of its residues. Furthermore, the DNA sequences of the frame-shift revertants discussed above predict relative isolectric point differences between the wild-type and various revertant forms of the polypeptide. The detection of these isolectric point differences by two-dimensional electrophoresis of subunit II from the various strains independently confirms the presumed reading frame of the gene. It is concluded that TGA is translated in yeast mitochondria, most probably as tryptophan.

Administration of the cytokines interferon-alpha and interleukin-2 is used for the treatment of various disorders, such as hepatitis C and various forms of cancer. The most serious side-effects are symptoms associated with depression, including fatigue, increased sleepiness, irritability, loss of appetite as well as cognitive changes. However, great differences exist in the prevalence of the development of depressive symptoms across studies. Differences in doses and duration of therapy may be sources of variation as well as individual differences of patients, such as a history of psychiatric illness. In addition, sensitization effects may contribute to differential responses of patients to the administration of cytokines. In animals administration of pro-inflammatory cytokines induces a pattern of behavioural alterations called 'sickness behaviour' which resembles the vegetative symptoms of depression in humans. Changes in serotonin (5-HT) receptors and in levels of 5-HT and its precursor tryptophan in depressed people support a role for 5-HT in the development of depression. In addition, evidence exists for a dysregulation of the noradrenergic system and a hyperactive hypothalamic-pituitary-adrenal (HPA) axis in depression. Some mechanisms exist which make it possible for cytokines to cross the blood-brain barrier. Pro-inflammatory cytokines such as IL-1beta, IFN-alpha, IFN-gamma and TNF-alpha affect the 5-HT metabolism directly and/or indirectly by stimulating the enzyme indoleamine 2,3-dioxygenase which leads to a peripheral depletion of tryptophan. IL-1, IL-2 and TNF-alpha influence noradrenergic activity and IL-1, IL-6 and TNF-alpha are found to be potent stimulators of the HPA axis. Altogether, administration of cytokines may induce alterations in the brain resembling those found in depressed patients, which leads to the hypothesis that cytokines induce depression by their influence on the 5-HT, noradrenergic and HPA system.

There is growing interest in the effects of antidepressant drug treatment on measures of emotional processing. Such actions may help us understand the role of monoamines in emotional dysfunction in depression and how antidepressant drug treatments work. Recent studies suggest that decreasing central serotonin function with tryptophan depletion can reinstate negative biases in recovered depressed patients, even at doses insufficient to induce changes in mood. Conversely, antidepressant drug administration increases the processing of positive emotional information in healthy volunteers and acutely depressed patients early in treatment. This increase in positive bias may provide a platform for subsequent cognitive restructuring and learning which contributes to the evolution of symptom change in depression. Functional neuroimaging studies suggest that these early antidepressant effects involve fronto-limbic and extra-striate circuitry suggestive of actions on both the initial appraisal and attentional processing of affective stimuli. This approach may therefore provide a framework for linking psychological and biological processes in emotional disorders and their treatment. Antidepressants may not directly modulate mood and anxiety but rather allow a different perspective for our ongoing evaluation of our self, the world and the future.

Genome-scale models (GEMs) of metabolism were constructed for 55 fully sequenced Escherichia coli and Shigella strains. The GEMs enable a systems approach to characterizing the pan and core metabolic capabilities of the E. coli species. The majority of pan metabolic content was found to consist of alternate catabolic pathways for unique nutrient sources. The GEMs were then used to systematically analyze growth capabilities in more than 650 different growth-supporting environments. The results show that unique strain-specific metabolic capabilities correspond to pathotypes and environmental niches. Twelve of the GEMs were used to predict growth on six differentiating nutrients, and the predictions were found to agree with 80% of experimental outcomes. Additionally, GEMs were used to predict strain-specific auxotrophies. Twelve of the strains modeled were predicted to be auxotrophic for vitamins niacin (vitamin B3), thiamin (vitamin B1), or folate (vitamin B9). Six of the strains modeled have lost biosynthetic pathways for essential amino acids methionine, tryptophan, or leucine. Genome-scale analysis of multiple strains of a species can thus be used to define the metabolic essence of a microbial species and delineate growth differences that shed light on the adaptation process to a particular microenvironment.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

Abnormalities in the kynurenine pathway may play a role in Huntington's disease (HD). In this study, tryptophan depletion and loading were used to investigate changes in blood kynurenine pathway metabolites, as well as markers of inflammation and oxidative stress in HD patients and healthy controls. Results showed that the kynurenine : tryptophan ratio was greater in HD than controls in the baseline state and after tryptophan depletion, indicating increased indoleamine dioxygenase activity in HD. Evidence for persistent inflammation in HD was provided by elevated baseline levels of C-reactive protein, neopterin and lipid peroxidation products compared with controls. The kynurenate : kynurenine ratio suggested lower kynurenine aminotransferase activity in patients and the higher levels of kynurenine in patients at baseline, after depletion and loading, do not result in any differences in kynurenic acid levels, providing no supportive evidence for a compensatory neuroprotective role for kynurenic acid. Quinolinic acid showed wide variations in blood levels. The lipid peroxidation data indicate a high level of oxidative stress in HD patients many years after disease onset. Levels of the free radical generators 3-hydroxykynurenine and 3-hydroxyanthranilic acid were decreased in HD patients, and hence did not appear to contribute to the oxidative stress. It is concluded that patients with HD exhibit abnormal handling of tryptophan metabolism and increased oxidative stress, and that these factors could contribute to ongoing brain dysfunction.

Immune tolerance is a central mechanism counteracting tumor-specific immunity and preventing effective anticancer immunotherapy. Induction of tolerance requires a specific environment in which tolerogenic dendritic cells (DCs) play an essential role deviating the immune response away from effective immunity. It was recently shown that maturation of DCs in the presence of PGE2 results in upregulation of indoleamine 2,3-dioxygenase (IDO) providing a potential mechanism for the development of DC-mediated Tcell tolerance. Here, we extend these findings, demonstrating a concomitant induction of IDO and secretion of soluble CD25 after DC maturation in the presence of PGE2. While maturation of DCs induced IDO expression on transcriptional level, only integration of PGE2 signaling led to up-regulation of functional IDO protein as well as significant expression of cell-surface and soluble CD25 protein. As a consequence, T-cell proliferation and cytokine production were significantly inhibited, which was mediated mainly by IDO-induced tryptophan depletion. Of importance, we demonstrate that different carcinoma entities associated with elevated levels of PGE2 coexpress CD25 and IDO in peritumoral dendritic cells, suggesting that PGE2 might influence IDO expression in human DCs in the tumor environment. We therefore suggest PGE2 to be a mediator of early events during induction of immune tolerance in cancer.