The Relationship Between Plasma Aß Levels, Cognitive Function and Brain Volumetrics: Sydney Memory and Ageing Study.

OBJECTIVES: Determine whether (1) a relationship exists between plasma amyloid-ß (Aß)1- 40 and 1-42 peptide levels, brain volumetrics and cognitive performance in elderly individuals with and without amnestic mild cognitive impairment (aMCI), (2) plasma Aß peptide levels differ between apolipoprotein E (APOE) ε4 carriers and non-carriers and (3) longitudinal changes in cognition and brain volume relate to Aß levels. METHODS: Subjects with aMCI (n = 89) and normal cognition (n = 126) were drawn from the Sydney Memory and Aging Study (Sydney MAS), a population based study of non-demented 70-90 year old individuals; 39 Alzheimer's disease (AD) patients were recruited from a specialty clinic. Sydney MAS participants underwent brain MRI scans and were assessed on 19 cognitive measures and were APOE ε4 genotyped. Plasma levels of Aß1-40 and 1-42 were quantified using ELISA. RESULTS: Wave1 plasma levels of Aß peptides and Aß1-42/1-40 ratio were lower in aMCI and AD, and Aß1-42 was positively associated with global cognition and hippocampal volume and negatively with white matter hyperintensities. The relationships of Aß1-40 and Aß1-42 were predominantly observed in ε4 allele carriers and non-carriers respectively. Longitudinal analysis revealed greater decline in global cognition and memory for the highest quintiles of Aß1-42 and the ratio measure. CONCLUSION: Plasma Aß levels and the Aß1-42/1-40 ratio are related to cognition and hippocampal volumes, with differential associations of Aß1-40 and Aß1-42 in ε4 carriers and non-carriers. These data support the Aß sink model of AD pathology, and suggest that plasma Aß measures may serve as biomarkers of AD.

Differential expression of sirtuins in the aging rat brain.

Although there are seven mammalian sirtuins (SIRT1-7), little is known about their expression in the aging brain. To characterize the change(s) in mRNA and protein expression of SIRT1-7 and their associated proteins in the brain of "physiologically" aged Wistar rats. We tested mRNA and protein expression levels of rat SIRT1-7, and the levels of associated proteins in the brain using RT-PCR and western blotting. Our data shows that SIRT1 expression increases with age, concurrently with increased acetylated p53 levels in all brain regions investigated. SIRT2 and FOXO3a protein levels increased only in the occipital lobe. SIRT3-5 expression declined significantly in the hippocampus and frontal lobe, associated with increases in superoxide and fatty acid oxidation levels, and acetylated CPS-1 protein expression, and a reduction in MnSOD level. While SIRT6 expression declines significantly with age acetylated H3K9 protein expression is increased throughout the brain. SIRT7 and Pol I protein expression increased in the frontal lobe. This study identifies previously unknown roles for sirtuins in regulating cellular homeostasis and healthy aging.

Upregulation of glycolytic enzymes, mitochondrial dysfunction and increased cytotoxicity in glial cells treated with Alzheimer's disease plasma.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each) from healthy controls, individuals with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD) on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ) found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia.

Plasma protein profiling of Mild Cognitive Impairment and Alzheimer's disease using iTRAQ quantitative proteomics.

BACKGROUND: With the promise of disease modifying treatments, there is a need for more specific diagnosis and prognosis of Alzheimer's disease (AD) and mild cognitive impairment (MCI). Plasma biomarkers are likely to be utilised to increase diagnostic accuracy and specificity of AD and cognitive decline. METHODS: Isobaric tags (iTRAQ) and proteomic methods were used to identify potential plasma biomarkers of MCI and AD. Relative protein expression level changes were quantified in plasma of 411 cognitively normal subjects, 19 AD patients and 261 MCI patients. Plasma was pooled into 4 groups including normal control, AD, amnestic single and multiple domain MCI (aMCI), and nonamnestic single and multiple domain MCI (nMCI). Western-blotting was used to validate iTRAQ data. Integrated function and protein interactions were explored using WEB based bioinformatics tools (DAVID v6.7 and STRING v9.0). RESULTS: In at least two iTRAQ replicate experiments, 30 proteins were significantly dysregulated in MCI and AD plasma, relative to controls. These proteins included ApoA1, ApoB100, complement C3, C4b-binding protein, afamin, vitamin D-binding protein precursor, isoform 1 of Gelsolin actin regulator, Ig mµ chain C region (IGHM), histidine-rich glycoprotein and fibrinogen ß and γ chains. Western-blotting confirmed that afamin was decreased and IGHM was increased in MCI and AD groups. Bioinformatics results indicated that these dysregulated proteins represented a diversity of biological processes, including acute inflammatory response, cholesterol transport and blood coagulation. CONCLUSION: These findings demonstrate that expression level changes in multiple proteins are observed in MCI and AD plasma. Some of these, such as afamin and IGHM, may be candidate biomarkers for AD and the predementia condition of MCI.

Mapping NAD(+) metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence.

Over the last decade, the importance of NAD(+) has expanded beyond its role as an essential cofactor for energy metabolism. NAD(+) has emerged as a major signalling molecule that serves as the sole substrate for several enzymatic reactions including the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), NAD-dependent protein deacetylases or CD38, and transcriptional factors by a new class of histone deacetylases known as sirtuins. NAD(+) levels are regulated by the metabolic status and cellular stress caused by oxidative stress and DNA damage. Since a detailed study of NAD(+) metabolism in the healthy ageing mammalian brain is nascent, we examined the effect of ageing on intracellular NAD(+) metabolism in different brain regions in female Wistar rats in young (3 months), middle aged (12 months) and older adults (24 months). Our results are the first to show a significant decline in intracellular NAD(+) levels and NAD:NADH ratio with ageing in the CNS, occurring in parallel to an increase in lipid peroxidation and protein oxidation (o- and m-tyrosine) and a decline in total antioxidant capacity. Hyperphosphorylation of H2AX levels was also observed together with increased PARP-1 and PARP-2 expression, and CD38 activity, concomitantly with reduced NAD(+) and ATP levels and SIRT1 function in the cortex, brainstem, hippocampus and cerebellum. Reduced activity of mitochondrial complex I-IV and impaired maximum mitochondrial respiration rate were also observed in the ageing rat brain. Among the multiple physiological pathways associated with NAD(+) catabolism, our discovery of CD38 as the major regulator of cellular NAD(+) levels in rat neurons indicates that CD38 is a promising therapeutic target for the treatment of age-related neurodegenerative diseases.

Plasma apolipoprotein levels are associated with cognitive status and decline in a community cohort of older individuals.

OBJECTIVES: Apolipoproteins have recently been implicated in the etiology of Alzheimer's disease (AD). In particular, Apolipoprotein J (ApoJ or clusterin) has been proposed as a biomarker of the disease at the pre-dementia stage. We examined a group of apolipoproteins, including ApoA1, ApoA2, ApoB, ApoC3, ApoE, ApoH and ApoJ, in the plasma of a longitudinal community based cohort. METHODS: 664 subjects (257 with Mild Cognitive Impairment [MCI] and 407 with normal cognition), mean age 78 years, from the Sydney Memory and Aging Study (MAS) were followed up over two years. Plasma apolipoprotein levels at baseline (Wave 1) were measured using a multiplex bead fluorescence immunoassay technique. RESULTS: At Wave 1, MCI subjects had lower levels of ApoA1, ApoA2 and ApoH, and higher levels of ApoE and ApoJ, and a higher ApoB/ApoA1 ratio. Carriers of the apolipoprotein E ε4 allele had significantly lower levels of plasma ApoE, ApoC3 and ApoH and a significantly higher level of ApoB. Global cognitive scores were correlated positively with ApoH and negatively with ApoJ levels. ApoJ and ApoE levels were correlated negatively with grey matter volume and positively with cerebrospinal fluid (CSF) volume on MRI. Lower ApoA1, ApoA2 and ApoH levels, and higher ApoB/ApoA1 ratio, increased the risk of cognitive decline over two years in cognitively normal individuals. ApoA1 was the most significant predictor of decline. These associations remained after statistically controlling for lipid profile. Higher ApoJ levels predicted white matter atrophy over two years. CONCLUSIONS: Elderly individuals with MCI have abnormal apolipoprotein levels, which are related to cognitive function and volumetric MRI measures cross-sectionally and are predictive of cognitive impairment in cognitively normal subjects. ApoA1, ApoH and ApoJ are potential plasma biomarkers of cognitive decline in non-demented elderly individuals.

M. tuberculosis induces potent activation of IDO-1, but this is not essential for the immunological control of infection.

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis.

Meta-analysis of plasma amyloid-ß levels in Alzheimer's disease.

Plasma amyloid-ß (Aß) levels have been proposed as biomarkers of Alzheimer's disease (AD), but studies have produced inconsistent results. We present a meta-analytic review of cross-sectional studies that examined plasma Aß levels in AD and cognitively normal subjects, and longitudinal studies that used baseline plasma Aß levels to predict conversion from normal cognition to AD. Medline and EMBASE databases were searched to generate an initial list of relevant studies, and selected authors approached for additional data. Twelve cross- sectional studies (n = 1483) and seven longitudinal (n = 3920) met the inclusion criteria for meta-analysis. Random effects model was used to calculate the weighted mean difference (WMD) by Review Manager Version 4.2. In longitudinal studies, cognitively normal individuals who converted to AD had higher baseline Aß1-40 and Aß1-42 levels (WMD: 10.29, z = 3.80, p = 0.0001 and WMD: 8.01, z = 2.76, p = 0.006, respectively), and non-significantly increased Aß1-42/Aß1-40 ratio (WMD: 0.03, z = 1.65, p = 0.10). In cross sectional studies, compared to cognitively normal individuals, AD patients had marginally but non-significantly lower Aß1-42 levels (WMD:-2.84, z = 1.73, p = 0.08), but Aß1-40 levels were not significantly different (WMD: 3.43, z = 0.40, p = 0.69). Our systematic review suggests a model of differential longitudinal changes in plasma Aß levels in cognitively stable individuals versus those who go on to develop AD dementia. Baseline Aß1-40 and Aß1-42 levels in cognitively normal elderly individuals might be predictors of higher rates of progression to AD, and should be further explored as potential biomarkers.

Chronic hepatitis C is associated with peripheral rather than hepatic insulin resistance.

BACKGROUND & AIMS: Chronic hepatitis C (CHC) is associated with insulin resistance (IR), liver steatosis (genotype 3), and increased diabetes risk. The site and mechanisms of IR are unclear. METHODS: We compared cross-sectionally 29 nonobese, normoglycemic males with CHC (genotypes 1 and 3) to 15 adiposity and age-matched controls using a 2-step hyperinsulinemic-euglycemic clamp with [6,6-(2)H(2)] glucose to assess insulin sensitivity in liver and peripheral tissues and (1)H-magnetic resonance spectroscopy to evaluate liver and intramyocellular lipid. Insulin secretion was assessed after intravenous glucose. RESULTS: Insulin secretion was not impaired in CHC. Peripheral insulin sensitivity was 35% higher in controls vs CHC (P < .001) during high-dose (264.3 +/- 25 [standard error] mU/L) insulin (P < .001); this was negatively associated with viral load (R(2) = .12; P = .05) and subcutaneous fat (R(2) = .41; P < .001). IR was similar in both genotypes despite 3-fold increased hepatic fat in genotype 3 (P < .001). Hepatic glucose production (P = .25) and nonesterified free fatty acid (P = .84) suppression with insulin were not different between CHC and controls inferring no adipocyte IR, and suggesting IR is mainly in muscle. In CHC, intramyocellular lipid was nonsignificantly increased but levels of glucagon (73.8 +/- 3.6 vs 52.8 +/- 3.1 ng/mL; P < .001), soluble tumor necrosis factor receptor 2 (3.1 +/- 0.1 vs 2.3 +/- 0.1 ng/mL; P < .001), and Lipocalin-2 (36.4 +/- 2.9 vs 19.6 +/- 1.6 ng/mL; P < .001) were elevated. CONCLUSIONS: CHC represents a unique infective/inflammatory model of IR, which is predominantly in muscle, correlates with subcutaneous, not visceral, adiposity, and is independent of liver fat.

Plasma biomarkers for mild cognitive impairment and Alzheimer's disease.

PURPOSE OF REVIEW: With the move toward development of disease modifying treatments, there is a need for more specific diagnosis of early Alzheimer's disease (AD) and mild cognitive impairment (MCI), plasma biomarkers are likely to play an important role in this. We review the current state of knowledge on plasma biomarkers for MCI and AD, including unbiased proteomics and very recent longitudinal studies. RECENT FINDINGS: With the use of proteomics methodologies, some proteins have been identified as potential biomarkers in plasma and serum of AD patients, including alpha-1-antitrypsin, complement factor H, alpha-2-macroglobulin, apolipoprotein J, apolipoprotein A-I. The findings of cross-sectional studies of plasma amyloid beta (A beta) levels are conflicting, but some recent longitudinal studies have shown that low plasma A beta 1-42 or A beta 1-40 levels, or A beta 1-42/A beta 1-40 ratio may be markers of cognitive decline. Other potential biomarkers for MCI and AD reflecting a variety of pathophysiological processes have been assessed, including isoprostanes and homocysteine (oxidative stress), total cholesterol and ApoE4 allele (lipoprotein metabolism), and cytokines and acute phase proteins (inflammation). A panel of 18 signal proteins was reported as markers of MCI and AD. SUMMARY: A variety of potential plasma biomarkers for AD and MCI have been identified, however the findings need replication in longitudinal studies. This area of research promises to yield interesting results in the near future.

Age and circadian influences on picolinic acid concentrations in human cerebrospinal fluid.

It has been suggested that picolinic acid (PIC), an endogenous metabolite of l-tryptophan, possesses neuro-protective and anti-proliferative effects within the CNS. However, the literature surrounding PIC is limited, and its exact endogenous function is not known. Picolinic acid is produced via the kynurenine pathway which has been implicated in the pathogenesis of a range of neuro-inflammatory diseases. Although not extensively studied, there have been reports of altered PIC production alongside other kynurenine metabolites in inflammatory disorders. In order to investigate whether PIC concentrations are altered with disease in the CNS, we analysed PIC levels in the CSF of 241 patients who underwent lumbar puncture as part of their standard clinical evaluation. In patients with no apparent CNS disease, CSF PIC levels were 10-fold higher in samples taken between 20:00 and 16:00 h compared with those collected between 04:00 and 12:00 h. This result suggests a diurnal variation in PIC synthesis within the CNS. In addition, we observed a direct correlation between a patient's age and their PIC concentration. No significant correlations were observed between CSF PIC levels and any specific disease state.

Mass spectrometry studies of the binding of the minor groove-directed alkylating agent alkamin to AT-tract oligonucleotides.

Minor groove binding alkylating agents, which have potential as cancer drugs, generate cytotoxic DNA adducts that are relatively resistant to repair as a consequence of locating covalent attachment at purine N3 nitrogen atoms. Recently, we used electrospray and matrix-assisted laser desorption ionization mass spectrometry to study the binding of the minor groove-directed polybenzamide bis-half-mustard alkamin, and its monofunctional analogue alkamini, to the oligonucleotide d(CGCGAATTCGCG)(2), identifying a number of inter- and intrastrand alkamin cross-links involving the GAATTC sequence [ Abdul Majid , A. M. S. , Smythe , G. , Denny , W. A. , and Wakelin , L. P. G. ( 2007 ) Mol. Pharmacol. 71 , 1165 - 1178 ]. Here, we extend these studies to d(CGCAAATTTGCG)(2), A3T3, and d(CGCAAAAAAGCG).d(CGCTTTTTTGCG), A6/T6, in which the opportunity for both inter- and intrastrand cross-linking is enhanced. We find that both ligands alkylate all adenines in the longer AT-tracts, as well as the abutting guanines, whether they are in the same strand as the adenines or not, in a manner consistent with covalent attack on purine N3 atoms from the minor groove. Alkamin forms intrastrand cross-links involving A4 and A6 and A6 and G10 in A3T3 and all of the purines in the A6/T6 purine tract, including G10. In addition, it forms interstrand cross-links between A4, A5, A6 and A4', A5', A6', between G10 and the latter adenines in A3T3, and between G22 and adenines A5 and A6 in A6/T6. The reactivity of the abutting guanines provides unexpected opportunities for both inter- and intrastrand cross-linking by alkamin, such as the interstrand cross-link in the CAAAAAAG sequence. We conclude that positioning monofunctional mustard groups on either end of a minor groove-directed polybenzamide has the capacity to enhance interstrand cross-links at all manner of AT-tracts, including most in which the adenines are all in one strand.

Microglial activation, macrophage infiltration, and evidence of cell death in the fetal brain after uteroplacental administration of lipopolysaccharide in sheep in late gestation.

OBJECTIVE: The purpose of this study was to determine whether uteroplacental delivery of endotoxin produces fetal systemic and central nervous system reactions that are suggestive of inflammation. STUDY DESIGN: Lipopolysaccharide (30 or 60 microg) was administered into the uterine artery of late gestation (135 +/- 0.3 days) pregnant sheep. Fetal blood was assayed to determine changes in levels of quinolinic acid, which is a metabolite of tryptophan that is produced by monocytes (macrophages, microglia). Fetal brains were collected after 72 hours and examined for the presence of activated microglia and parenchymal macrophages. RESULTS: The brains of treated fetuses showed microglial activation and macrophage infiltration, which varied between brain region and lipopolysaccharide dose. Cell death that had been determined by cresyl violet/acid fuchsin staining was observed in the external capsule. There was significant increase of quinolinic acid in the fetal circulation, but no lipopolysaccharide was detected. CONCLUSION: Uteroplacental inflammation results in significant microglial activation and macrophage infiltration without direct fetal exposure to endotoxin, which suggests that placental responses contribute to perinatal brain damage that is associated with infection during pregnancy.

Kynurenine pathway metabolism in human blood-brain-barrier cells: implications for immune tolerance and neurotoxicity.

The catabolic pathway of l-tryptophan (l-trp), known as the kynurenine pathway (KP), has been implicated in the pathogenesis of a wide range of brain diseases through its ability to lead to immune tolerance and neurotoxicity. As endothelial cells (ECs) and pericytes of the blood-brain-barrier (BBB) are among the first brain-associated cells that a blood-borne pathogen would encounter, we sought to determine their expression of the KP. Using RT-PCR and HPLC/GC-MS, we show that BBB ECs and pericytes constitutively express components of the KP. BBB ECs constitutively synthesized kynurenic acid, and after immune activation, kynurenine (KYN), which is secreted basolaterally. BBB pericytes produced small amounts of picolinic acid and after immune activation, KYN. These results have significant implications for the pathogenesis of inflammatory brain diseases in general, particularly human immunodeficiency virus (HIV)-related brain disease. Kynurenine pathway activation at the BBB results in local immune tolerance and neurotoxicity: the basolateral secretion of excess KYN can be further metabolized by perivascular macrophages and microglia with synthesis of quinolinic acid. The results point to a mechanism whereby a systemic inflammatory signal can be transduced across an intact BBB to cause local neurotoxicity.

Characterization of the kynurenine pathway in human neurons.

The kynurenine pathway is a major route of L-tryptophan catabolism producing neuroactive metabolites implicated in neurodegeneration and immune tolerance. We characterized the kynurenine pathway in human neurons and the human SK-N-SH neuroblastoma cell line and found that the kynurenine pathway enzymes were variably expressed. Picolinic carboxylase was expressed only in primary and some adult neurons but not in SK-N-SH cells. Because of this difference, SK-N-SH cells were able to produce the excitotoxin quinolinic acid, whereas human neurons produced the neuroprotectant picolinic acid. The net result of kynurenine pathway induction in human neurons is therefore predicted to result in neuroprotection, immune regulation, and tumor inhibition, whereas in SK-N-SH cells, it may result in neurotoxicity, immune tolerance, and tumor promotion. This study represents the first comprehensive characterization of the kynurenine pathway in neurons and the first description of the involvement of the kynurenine pathway as a mechanism for controlling both tumor cell neurotoxicity and persistence.

Quantitative determination of ortho- and meta-tyrosine as biomarkers of protein oxidative damage in beta-thalassemia.

Oxidative stress in thalassemia is caused by secondary iron overload and stems from blood transfusion and increased iron uptake. In this study, we hypothesized that levels of o- and m-tyrosine, products of hydroxyl radical attack on phenylalanine, would be elevated in beta-thalassemia (intermediate). This study represents the first report in which specific markers of protein oxidative damage have been quantified in thalassemia. We used GC/MS to assay o- and m-tyrosine at the femtomole level using only a few microliters of plasma. Levels of both markers were significantly higher in patients with beta-thalassemia than in controls and were positively correlated with serum ferritin, malondialdehyde, superoxide dismutase, glutathione peroxidase and glutathione. We conclude that o- and m-tyrosine are useful biomarkers of oxidative damage to proteins in thalassemia (intermediate) and may also be useful markers in other iron overload diseases. Positive correlations between o- and m-tyrosine levels and malondialdehyde as well as antioxidants such as superoxide dismutase, glutathione peroxidase and glutathione, are indicative of the broad impact of oxidative stress on blood plasma in thalassemia, with up-regulation of antioxidant proteins probably reflecting a homeostatic response to these increased stress levels.

Dynorphin knockout reduces fat mass and increases weight loss during fasting in mice.

Endogenous opioids, particularly dynorphins, have been implicated in regulation of energy balance, but it is not known how they mediate this in vivo. We investigated energy homeostasis in dynorphin knockout mice (Dyn(-/-) mice) and probed the interactions between dynorphins and the neuropeptide Y (NPY) system. Dyn(-/-) mice were no different from wild types with regards to body weight and basal and fasting-induced food intake, but fecal output was increased, suggesting decreased nutrient absorption, and they had significantly less white fat and lost more weight during a 24-h fast. The neuroendocrine and thermal responses to fasting were at least as pronounced in Dyn(-/-) as in wild types, and there was no stimulatory effect of dynorphin knockout on 24-h energy expenditure (kilocalories of heat produced) or physical activity. However, Dyn(-/-) mice showed increased circulating concentrations of 3,4-dihydroxyphenlacetic acid and 3,4-dihydroxyphenylglycol, suggesting increased activity of the sympathetic nervous system. The respiratory exchange ratio of male but not female Dyn(-/-) mice was reduced, demonstrating increased fat oxidation. Interestingly, expression of the orexigenic acting NPY in the hypothalamic arcuate nucleus was reduced in Dyn(-/-) mice. However, fasting-induced increases in pre-prodynorphin expression in the arcuate nucleus, the paraventricular nucleus, and the ventromedial hypothalamus but not the lateral hypothalamus were abolished by deletion of Y(1) but not Y(2) receptors. Therefore, ablation of dynorphins results in increases in fatty acid oxidation in male mice, reductions in adiposity, and increased weight loss during fasting, possibly via increases in sympathetic activity, decreases in intestinal nutrient absorption, and interactions with the NPYergic system.

Structure of the d(CGCGAATTCGCG)2 complex of the minor groove binding alkylating agent alkamin studied by mass spectrometry.

Nitrogen mustard alkylating agents are important cancer drugs. Much interest has been focused on redirecting their covalent adducts from the N7 atoms of guanine in the major groove of DNA to the N3 atoms of adenine in the minor groove by attaching mustard groups to AT-selective minor groove binding ligands. Here we describe the use of electrospray ionization and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry to study the structure of the DNA complexes of two minor groove binding polybenzamide mustards, alkamin and alkamini; the former is a bis-half-mustard in which reactive groups are disposed at each end of the ligand, and the latter is its monofunctional analog. Alkamin is potently cytotoxic and active in experimental mouse tumor models, whereas alkamini is not. We have studied their interaction with the DNA dodecamer d(CGCGAATTCGCG)(2), designated A2T2, and we provide a detailed analysis of the observed DNA-ligand adduct ions and their fragmentation products. We find that alkamini alkylates A2T2 at guanine G4 and adenines A5 and A6 in a manner consistent with covalent attack on purine N3 atoms from the minor groove of the AT tract. Alkamin also forms monofunctional adducts at G4 and both adenines in which the second mustard arm is hydrolyzed but, in addition, forms a variety of interstrand cross-links between adenines A5/A6 and A5'/A6', an interstrand cross-link between G4 and A6', and an intrastrand cross-link between G4 and A6. We conclude that the marked cytotoxicity of alkamin and its experimental antitumor activity could be the consequence of its ability to cross-link cellular DNA at AT tract sequences.

Inhibition of indoleamine 2,3 dioxygenase activity by H2O2.

Indoleamine 2,3-dioxygenase is the first and rate limiting enzyme of the kynurenine pathway of tryptophan metabolism, has potent effects on cell proliferation and mediates antimicrobial, antitumorogenic, and immunosuppressive effects. As a potent cytotoxic effector, the mechanisms of indoleamine 2,3-dioxygenase inhibition deserve greater attention. The work presented here represents the first systematic study exploring the mechanisms by which low levels of hydrogen peroxide (10-100 microM) inhibit indoleamine 2,3-dioxygenase in vitro. Following brief peroxide exposure both enzyme inhibition and structural changes were observed. Loss of catalysis was accompanied by oxidation of several cysteine residues to sulfinic and sulfonic acids, observed by electrospray and MALDI mass spectrometry. Enzyme activity could in part be preserved in the presence of sulfhydryl containing compounds, particularly DTT and methionine. However, these structural alterations did not prevent substrate (l-tryptophan) binding. Some enzyme activity could be recovered in the presence of thioredoxin, indicating that the inhibitory effect of H(2)O(2) is at least partially reversible in vitro. We present evidence that cysteine oxidation represents one mechanism of indoleamine 2,3-dioxygenase inhibition.

Prolonged survival of a murine model of cerebral malaria by kynurenine pathway inhibition.

C57BL/6J mice infected with Plasmodium berghei ANKA develop neurological dysfunction and die within 7 days of infection. We show that treatment of infected mice with a kynurenine-3-hydroxylase inhibitor prevents them from developing neurological symptoms and extends their life span threefold until severe anemia develops.