Human and Mouse Type I Natural Killer T Cell Antigen Receptors Exhibit Different Fine Specificities for CD1d-Antigen Complex

Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4′,4″-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4′-OH position in comparison with the 3′-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4′-OH > 3′-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.     

Mitochondrial DNA sequence variation is associated with free-living activity energy expenditure in the elderly

The decline in activity energy expenditure underlies a range of age-associated pathological conditions, neuromuscular and neurological impairments, disability, and mortality. The majority (90%) of the energy needs of the human body are met by mitochondrial oxidative phosphorylation (OXPHOS). OXPHOS is dependent on the coordinated expression and interaction of genes encoded in the nuclear and mitochondrial genomes. We examined the role of mitochondrial genomic variation in free-living activity energy expenditure (AEE) and physical activity levels (PAL) by sequencing the entire (~16.5 kilobases) mtDNA from 138 Health, Aging, and Body Composition Study participants. Among the common mtDNA variants, the hypervariable region 2 m.185G>A variant was significantly associated with AEE (p=0.001) and PAL (p=0.0005) after adjustment for multiple comparisons. Several unique nonsynonymous variants were identified in the extremes of AEE with some occurring at highly conserved sites predicted to affect protein structure and function. Of interest is the p.T194M, CytB substitution in the lower extreme of AEE occurring at a residue in the Qi site of complex III. Among participants with low activity levels, the burden of singleton variants was 30% higher across the entire mtDNA and OXPHOS complex I when compared to those having moderate to high activity levels. A significant pooled variant association across the hypervariable 2 region was observed for AEE and PAL. These results suggest that mtDNA variation is associated with free-living AEE in older persons and may generate new hypotheses by which specific mtDNA complexes, genes, and variants may contribute to the maintenance of activity levels in late life.     

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O(2)(-)) and nitric oxide (NO) forms peroxynitrite (ONOO(-)) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO(-). Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O(2)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO(-). We also found that ONOO(-) directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO(-) formed oligomers. Resveratrol (RES), a scavenger of O(2)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) and ONOO(-) levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO(-) during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.     

Stimulus and network dynamics collide in a ratiometric model of the antennal lobe macroglomerular complex

Time is considered to be an important encoding dimension in olfaction, as neural populations generate odour-specific spatiotemporal responses to constant stimuli. However, during pheromone mediated anemotactic search insects must discriminate specific ratios of blend components from rapidly time varying input. The dynamics intrinsic to olfactory processing and those of naturalistic stimuli can therefore potentially collide, thereby confounding ratiometric information. In this paper we use a computational model of the macroglomerular complex of the insect antennal lobe to study the impact on ratiometric information of this potential collision between network and stimulus dynamics. We show that the model exhibits two different dynamical regimes depending upon the connectivity pattern between inhibitory interneurons (that we refer to as fixed point attractor and limit cycle attractor), which both generate ratio-specific trajectories in the projection neuron output population that are reminiscent of temporal patterning and periodic hyperpolarisation observed in olfactory antennal lobe neurons. We compare the performance of the two corresponding population codes for reporting ratiometric blend information to higher centres of the insect brain. Our key finding is that whilst the dynamically rich limit cycle attractor spatiotemporal code is faster and more efficient in transmitting blend information under certain conditions it is also more prone to interference between network and stimulus dynamics, thus degrading ratiometric information under naturalistic input conditions. Our results suggest that rich intrinsically generated network dynamics can provide a powerful means of encoding multidimensional stimuli with high accuracy and efficiency, but only when isolated from stimulus dynamics. This interference between temporal dynamics of the stimulus and temporal patterns of neural activity constitutes a real challenge that must be successfully solved by the nervous system when faced with naturalistic input.     

Atorvastatin activates heme oxygenase-1 at the stress response elements

Statins are known to inhibit growth of a number of cancer cells, but their mechanism of action is not well established. In this study, human prostate adenocarcinoma PC-3 and breast adenocarcinoma MCF-7 cell lines were used as models to investigate the mechanism of action of atorvastatin, one of the statins. Atorvastatin was found to induce apoptosis in PC-3 cells at a concentration of 1 μM, and in MCF-7 cells at 50 μM. Initial survey of possible pathway using various pathway-specific luciferase reporter assays showed that atorvastatin-activated antioxidant response element (ARE), suggesting oxidative stress pathway may play a role in atorvastatin-induced apoptosis in both cell lines. Among the antioxidant response genes, heme oxygenase-1 (HO-1) was significantly up-regulated by atorvastatin. Pre-incubation of the cells with geranylgeranyl pyrophosphate blocked atorvastatin-induced apoptosis, but not up-regulation of HO-1, suggesting that atorvastatin-induced apoptosis is dependent on GTPase activity and up-regulation of HO-1 gene is not. Six ARE-like elements (designated StRE1 [stress response element] through StRE6) are present in the HO-1 promoter. Atorvastatin was able to activate all of the elements. Because these StRE sites are present in clusters in HO-1 promoter, up-regulation of HO-1 by atorvastatin may involve multiple StRE sites. The role of HO-1 in atorvastatin-induced apoptosis in PC-3 and MCF-7 remains to be studied.     

An apolipoprotein A-V gene SNP is associated with marked hypertriglyceridemia among Asian-American patients

Apolipoprotein A-V (apoA-V) is an important regulator of plasma levels of triglyceride (TG) in mice. In humans, APOA5 genetic variation is associated with TG in several populations. In this study, we determined the effects of the p.185Gly>Cys (c.553G>T; rs2075291) polymorphism on plasma TG levels in subjects of Chinese ancestry living in the United States and in a group of non-Chinese Asian ancestry. The frequency of the less common cysteine allele was 4-fold higher (15.1% vs. 3.7%) in Chinese high-TG subjects compared with a low-TG group (Chi-square = 20.2; P < 0.0001), corresponding with a 4.45 times higher risk of hypertriglyceridemia (95% confidence interval, 2.18-9.07; P < 0.001). These results were replicated in the non-Chinese Asians. Heterozygosity was associated, in the high-TG group, with a doubling of TG (P < 0.001), mainly VLDL TG (P = 0.014). All eleven TT homozygotes had severe hypertriglyceridemia, with mean TG of 2,292 +/- 447 mg/dl. Compared with controls, carriers of the T allele had lower postheparin lipoprotein lipase activity but not hepatic lipase activity. In Asian populations, this common polymorphism can lead to profound adverse effects on lipoprotein profiles, with homozygosity accounting for a significant number of cases of severe hypertriglyceridemia. This specific apoA-V variant has a pronounced effect on TG metabolism, the mechanism of which remains to be elucidated.     

Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase-ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long-term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial-derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up-regulated by 4-fold 7 days after injury and gradually decreased to normal at 28 days post-lesion. In chondroitinase-treated animals, the total GAG remained at low level throughout the 28-day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.     

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.     

Familial idiopathic basal ganglia calcification (Fahr's disease) without neurological, cognitive and psychiatric symptoms is not linked to the IBGC1 locus on chromosome 14q

Idiopathic basal ganglia calcification (IBGC) is characterised by radiological, neurological, cognitive and psychiatric abnormalities. The associations between these abnormal phenotypes and abnormal genes remain unclear despite the recent mapping to chromosome 14q of a susceptibility locus for IBGC ( IBGC1). We identified two siblings, from a large multigenerational pedigree, who had both been diagnosed with radiological IBGC, dementia, bipolar affective disorder and Parkinsonism. We assessed (1) other family members to determine whether these four phenotypes were co-segregating as symptoms of IBGC, and (2) possible IBGC linkage to the IBGC1 locus on chromosome 14q or to any known or potential dementia genes. Nine second-generation and 21 third-generation members received radiological, neurological, neuropsychological and psychiatric assessments. We genotyped all family members for microsatellite markers at the IBGC1 locus and polymorphisms of the ApoE, VLDL, alpha1-ACT, BChE-K, APP, PS1, PS2 and tau genes and tested these for linkage to IBGC, dementia and bipolar disorder. Of the ten family members with radiological intracranial calcification, all except the two index cases were normal. There was no significant association between IBGC status and severe cognitive impairment or dementia ( P=0.335) or bipolar affective disorder or Parkinsonism ( P=1.0). Linkage to the IBGC1 locus was excluded. Of the eight dementia gene markers tested, the only positive LOD score was for the ApoE epsilon4 polymorphism and dementia/severe cognitive impairment. We have identified a form of IBGC in which calcification is inherited independently of neurological, cognitive and psychiatric symptoms. This may represent a second locus for this disorder.     

Molecular cloning, expression, localization, and gene organization of PTX1, a human nuclear protein that is downregulated in prostate cancer.

A cDNA, designated PTX1, has been isolated by subtractive hybridization on the basis that it is expressed in normal prostate but not in prostate carcinoma. The full-length cDNA was subsequently established by 5' and 3' RACE. Nucleotide sequence analysis of the 5'- and 3'-RACE clones yielded a composite cDNA of 1327 bp, which predicted a protein of 377 amino acid residues with a putative nuclear import signal (RRLNRKK) at its N terminus. The PTX1 gene was localized to human chromosome 12 and was found to be ubiquitously expressed. A segment of the cDNA was expressed in E. coli to produce a fragment of the PTX1 protein for the generation of specific antibodies. The resulting antibodies detected a 73-kDa protein in both nuclear and cytoplasmic extracts of prostate, although the level in the cytoplasmic extract was much lower. Using immunohistochemical analysis, the PTX1 protein was localized mainly in the nuclei of glandular epithelia of normal prostate. The nuclear staining was greatly reduced in prostate carcinoma. The gene organization of PTX1 was established by comparing the cDNA sequence with the published human genomic sequence.