Cerebral [18 F]T807/AV1451 retention pattern in clinically probable CTE resembles pathognomonic distribution of CTE tauopathy.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder most commonly associated with repetitive traumatic brain injury (TBI) and characterized by the presence of neurofibrillary tangles of tau protein, known as a tauopathy. Currently, the diagnosis of CTE can only be definitively established postmortem. However, a new positron emission tomography (PET) ligand, [18F]T807/AV1451, may provide the antemortem detection of tau aggregates, and thus various tauopathies, including CTE. Our goal was to examine [18F]T807/AV1451 retention in athletes with neuropsychiatric symptoms associated with a history of multiple concussions. Here we report a 39-year-old retired National Football League player who suffered 22 concussions and manifested progressive neuropsychiatric symptoms. Emotional lability and irritability were the chief complaints. Serial neuropsychological exams revealed a decline in executive functioning, processing speed and fine motor skills. Naming was below average but other cognitive functions were preserved. Structural analysis of longitudinally acquired magenetic resonance imaging scans revealed cortical thinning in the left frontal and lateral temporal areas, as well as volume loss in the basal ganglia. PET with [18F]florbetapir was negative for amyloidosis. The [18F]T807/AV1451 PET showed multifocal areas of retention at the cortical gray matter-white matter junction, a distribution considered pathognomonic for CTE. [18F]T807/AV1451 standard uptake value (SUV) analysis showed increased uptake (SUVr⩾1.1) in bilateral cingulate, occipital, and orbitofrontal cortices, and several temporal areas. Although definitive identification of the neuropathological underpinnings basis for [18F]T807/AV1451 retention requires postmortem correlation, our data suggest that [18F]T807/AV1451 tauopathy imaging may be a promising tool to detect and diagnose CTE-related tauopathy in living subjects.

Interactive effects of age and estrogen on cortical neurons: implications for cognitive aging.

In the past few decades it has become clear that estrogen signaling plays a much larger role in modulating the cognitive centers of the brain than previously thought possible. We have developed a nonhuman primate (NHP) model to investigate the relationships between estradiol (E) and cognitive aging. Our studies of cyclical E treatment in ovariectomized (OVX) young and aged rhesus monkeys have revealed compelling cognitive and synaptic effects of E in the context of aging. Delayed response (DR), a task that is particularly dependent on integrity of dorsolateral prefrontal cortex (dlPFC) area 46 revealed the following: (1) that young OVX rhesus monkeys perform equally well whether treated with E or vehicle (V), and (2) that aged OVX animals given E perform as well as young adults with or without E, whereas OVX V-treated aged animals display significant DR impairment. We have analyzed the structure of layer III pyramidal cells in area 46 in these same monkeys. We found both age and treatment effects on these neurons that are consistent with behavioral data. Briefly, reconstructions of pyramidal neurons in area 46 from these monkeys showed that cyclical E increased the density of small, thin spines in both young and aged monkeys. However, this effect of E was against a background of age-related loss of small, thin spines, leaving aged V-treated monkeys with a particularly low density of these highly plastic spines, and vulnerable to cognitive decline. Our current interpretation is that E not only plays a critically important role in maintaining spine number, but also enables synaptic plasticity through a cyclical increase in small highly plastic spines that may be stabilized in the context of learning. Interestingly, recent studies demonstrate that chronic E is less effective at inducing spinogenesis than cyclical E. We have begun to link certain molecular attributes of excitatory synapses in area 46 to E effects and cognitive performance in these monkeys. Given the importance of synaptic estrogen receptor α (ER-α) in rat hippocampus, we focused our initial studies on synaptic ER-α in area 46. Three key findings have emerged from these studies: (1) synaptic ER-α is present in axospinous synapses in area 46; (2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and (3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age and treatment groups. These findings have important implications for the design of hormone treatment strategies for both surgically and naturally menopausal women. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.

Stereologic analysis of neurofibrillary tangle formation in prefrontal cortex area 9 in aging and Alzheimer's disease.

Alzheimer's disease (AD) is characterized neuropathologically by several features including extensive neuronal death in the cerebral cortex. In fact, while neuropathological changes restricted to the hippocampal formation are a consistent reflection of age-related memory impairment, overt dementia is present only in cases with neocortical involvement. Several quantitative studies have reported a substantial loss of neurons from these regions and a parallel increase in the number of neurofibrillary tangles (NFT). However, accurate quantitative data on the dynamics of NFT formation are lacking. In the present study, we performed a stereologic analysis of the proportions of intracellular and extracellular (ghost) NFT, and unaffected neurons in the deep part of layer III (layer IIIc) and the superficial part of layer V (layer Va) of Brodmann's prefrontal cortex area 9. Elderly cognitively unimpaired cases were compared with cases with different degrees of cognitive dysfunction. The data revealed differential rates of formation of intracellular and extracellular NFT between the two layers, and confirmed the presence of a severe disease-associated, but not age-related, neuronal loss. It was also shown that a susbtantial number of pyramidal cells may persist either unaffected or in a transitional stage of NFT formation in both neocortical layers. These results suggest that a considerable number of neurons containing an intracellular NFT exists in the neocortex until late in the course of AD. Whereas it is not possible to assess whether such transitional neurons are fully functional, these affected neurons might respond positively to therapeutic strategies aimed at protecting the cells that are prone to neurofibrillary degeneration in AD.

Neuronal subclass-selective loss of pyruvate dehydrogenase immunoreactivity following canine cardiac arrest and resuscitation.

Chronic impairment of aerobic energy metabolism accompanies global cerebral ischemia and reperfusion and likely contributes to delayed neuronal cell death. Reperfusion-dependent inhibition of pyruvate dehydrogenase complex (PDHC) enzyme activity has been described and proposed to be at least partially responsible for this metabolic abnormality. This study tested the hypothesis that global cerebral ischemia and reperfusion results in the loss of pyruvate dehydrogenase immunoreactivity and that such loss is associated with selective neuronal vulnerability to transient ischemia. Following 10 min canine cardiac arrest, resuscitation, and 2 or 24 h of restoration of spontaneous circulation, brains were either perfusion fixed for immunohistochemical analyses or biopsy samples were removed for Western immunoblot analyses of PDHC immunoreactivity. A significant decrease in immunoreactivity was observed in frontal cortex homogenates from both 2 and 24 h reperfused animals compared to samples from nonischemic control animals. These results were supported by confocal microscopic immunohistochemical determinations of pyruvate dehydrogenase immunoreactivity in the neuronal cell bodies located within different layers of the frontal cortex. Loss of immunoreactivity was greatest for pyramidal neurons located in layer V compared to neurons in layers IIIc/IV, which correlates with a greater vulnerability of layer V neurons to delayed death caused by transient global cerebral ischemia.

Differential distribution of presenilin-1, Bax, and Bcl-X(L) in Alzheimer's disease and frontotemporal dementia.

We have previously reported that presenilin-1 (PS-1)-immunoreactive neurons survive in late-onset sporadic Alzheimer's disease (AD). To examine if this is also the case in other dementing conditions, and if it is associated with changes in the expression of the main apoptosis-related proteins, a quantitative immunocytochemical study of presenilin-1, Bax, and Bcl-X(L) in the cerebral cortex of non-demented and AD patients, and patients with frontotemporal dementia (FTD) was performed. In non-demented cases, the frequency of neurons showing PS-1 immunoreactivity was 25-60%, Bax immunoreactivity 36-54%, and Bcl-X(L) immunoreactivity 26-63% depending on the cortical area. The frequency of NFT-free neurons which contained PS-1 or Bax was consistently increased in all of the areas in AD. In FTD cases, the percentage of PS-I-, but not Bax-immunoreactive neurons was increased only in areas displaying a substantial neuronal loss. Conversely, there was no difference in the densities of Bcl-X(L)-containing neurons among the three diagnosis groups. These data suggest that surviving neurons in affected cortical areas in AD show a high expression of PS-1 and Bax, indicating that these proteins play a key role in the mechanisms of cell death in this disorder. In FTD, neurons containing PS-1 are preserved, further supporting a neuroprotective role for this protein in other neurodegenerative disorders.

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain.

Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actin-fragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 A resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity.

Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration.

Neuronal inclusions with bundles of abnormal filaments made of tau polymers are found in numerous diseases with neurofibrillary degeneration. Tau proteins are the basic components of paired helical filaments (PHF) in Alzheimer's disease (AD), and are abnormally phosphorylated. A disease-specific phosphorylation site at serine422 was demonstrated on PHF, but not on tau proteins from biopsy-derived brain samples. In the present study, we report the characterization of a polyclonal antibody (988) against the serine422 phosphorylation site. By using biochemical and immunohistochemical methods, we confirmed that it is not found on tau proteins from biopsy- or autopsy-derived control samples, and we investigated the presence of this epitope on tau proteins in several neurodegenerative disorders, including AD, Down syndrome (DS), Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), postencephalitic parkinsonism (PEP) and Pick's disease (PiD). By Western blotting, antibody 988 labeled the characteristic tau triplet (tau 55, 64, 69) in AD, DS, Guamanian ALS/PDC and PEP. PSP and CBD exhibited their typical tau doublet (tau 64, 69), whereas the doublet tau 55 and 64 was detected in PiD. In all of these neurodegenerative disorders, antibody 988 clearly labeled NFT and dystrophic neurites, as well as Pick bodies in PiD cases, whereas no staining was observed in control cases. These data indicate that phosphorylation of serine422 on tau proteins is a common feature among neurodegenerative disorders and is therefore not specific of AD. Moreover, phosphorylation of this epitope permits the distinction between normal tau proteins and pathological tau proteins.

Altered specificity mutations define residues essential for substrate positioning in xanthine dehydrogenase.

We describe the sequence changes of a number of mutations of the Aspergillus nidulans xanthine dehydrogenase (XDH). We have located the amino acids affected by these changes in the three-dimensional (3D) structure of aldehyde oxido-reductase (MOP) from Desulfovibrio gigas, related to eukaryotic XDHs. Of these, two are loss of function mutations, mapping, respectively, in the molybdenum-pterin co-factor (MoCo) domain and in the domain involved in substrate recognition. Changes in two amino acids result in resistance to the irreversible inhibitor allopurinol. In Arg911 two different changes, conserved among all XDHs and MOP but not in other aldehyde oxidases (AO), change the position of hydroxylation of the analogue 2-hydroxypurine from C-8 to C-6. A number of changes affect residues adjacent to the molybdenum or its ligands. Arg911 is positioned in the substrate pocket in a way that it can account for the positioning of purine substrates in relation to the MoCo reactive center, together with a glutamate residue, universally conserved among the XDHs (Glu833).

Crystal structure of the tyrosine phosphatase SHP-2.

The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains. In the absence of a tyrosine-phosphorylated binding partner, the N-terminal SH2 domain binds the phosphatase domain and directly blocks its active site. This interaction alters the structure of the N-SH2 domain, disrupting its phosphopeptide-binding cleft. Conversely, interaction of the N-SH2 domain with phosphopeptide disrupts its phosphatase recognition surface. Thus, the N-SH2 domain is a conformational switch; it either binds and inhibits the phosphatase, or it binds phosphoproteins and activates the enzyme. Recognition of bisphosphorylated ligands by the tandem SH2 domains is an integral element of this switch; the C-terminal SH2 domain contributes binding energy and specificity, but it does not have a direct role in activation.

Receptors for advanced glycosylation endproducts in human brain: role in brain homeostasis.

BACKGROUND: Advanced glycation end products (AGEs) are the reactive derivatives of nonenzymatic glucose-macromolecule condensation products. Aging human tissues accumulate AGEs in an age-dependent manner and contribute to age-related functional changes in vital organs. We have shown previously that AGE scavenger receptors are present on monocyte/macrophages, lymphocytes, and other cells. However, it remains unclear whether the human brain can efficiently eliminate AGE-modified proteins and whether excessive AGEs can contribute to inflammatory changes leading to brain injury in aging. MATERIALS AND METHODS: To explore the expression and characteristics of AGE-binding proteins on CNS glia components and their putative function, such as degradation of AGE-modified proteins, primary human astrocytes and human monocytes (as a microglial cell surrogate) and murine microglia (N9) cells and cell membrane extracts were used. Immunohistochemistry was used to examine the distribution of AGE-binding proteins in the human hippocampus; RT-PCR techniques were used to examine the biologic effects of AGEs and a model AGE compound, FFI, on AGE-binding protein modulation and cytokine responses of human astrocytes and monocytes. RESULTS: Our results showed that AGE-binding proteins AGE-R1, -R2, and -R3 are present in glial cells. Western blot analyses and radiolabeled ligand binding studies show that AGE-R1 and -R3 from human astrocytes bind AGE-modified proteins; binding could be blocked by anti-AGE-R1 and anti-AGE-R3 antibodies, respectively. Immunohistochemistry showed that AGE-R1 and -R2 are expressed mainly in neurons; only some glial cells express these AGE-binding proteins. In contrast, AGE-R3 was found only on those astrocytes whose positively stained foot processes extend and surround the sheath of microcapillaries. RT-PCR results showed that mRNAs of the three AGE-binding proteins are expressed constitutively in human astrocytes and monocytes, and receptor transcripts are not regulated by exogenous AGEs, the model AGE compound FFI, or phorbol ester. At the concentrations used, GM-CSF appears to be the only cytokine whose transcript and protein levels are regulated in human astrocytes by exogenous AGEs. CONCLUSIONS: The selective presence of AGE-binding proteins in pyramidal neurons and glial cells and their roles in degrading AGE-modified protein in glial cells suggest that the human brain has a mechanism(s) to clear AGE-modified proteins. Without this capacity, accumulation of AGEs extracellularly could stimulate glial cells to produce the major inflammatory cytokine GM-CSF, which has been shown to be capable of up-regulating AGE-R3. It remains to be determined whether AGE-binding proteins could be aberrant or down-regulated under certain pathological conditions, resulting in an insidious inflammatory state of the CNS in some aging humans.

Life and death of neurons in the aging brain.

Neurodegenerative disorders are characterized by extensive neuron death that leads to functional decline, but the neurobiological correlates of functional decline in normal aging are less well defined. For decades, it has been a commonly held notion that widespread neuron death in the neocortex and hippocampus is an inevitable concomitant of brain aging, but recent quantitative studies suggest that neuron death is restricted in normal aging and unlikely to account for age-related impairment of neocortical and hippocampal functions. In this article, the qualitative and quantitative differences between aging and Alzheimer's disease with respect to neuron loss are discussed, and age-related changes in functional and biochemical attributes of hippocampal circuits that might mediate functional decline in the absence of neuron death are explored. When these data are viewed comprehensively, it appears that the primary neurobiological substrates for functional impairment in aging differ in important ways from those in neurodegenerative disorders such as Alzheimer's disease.

Thermus thermophilus cytochrome-c552: A new highly thermostable cytochrome-c structure obtained by MAD phasing.

The three-dimensional structure of cytochrome-c552 from Thermus thermophilus has been determined by the multiple anomalous dispersion technique using synchrotron radiation and refined to a resolution of 1.28 A. Data collection at 90 K and the recording of three data sets (f'-minimum: 7125 eV, f"-maximum: 7138 eV and reference for scaling: 10,077 eV) resulted in an initial electron density of very high quality at 2.1 A, which was readily interpretable for model building. The model was refined to an R value of 19.1% (Rfree=22.4%) at 1.28 A resolution using a fourth data set collected at a photon energy of 11,810 eV. Comparison of this thermophilic cytochrome with its mesophilic mitochondrial or bacterial counterparts reveals significant structural differences which are discussed with respect to their importance for thermostability and binding between this cytochrome and its corresponding ba3-oxidase. Amino acid sequence similarities to other class I cytochromes are very weak and entirely limited to the region around the CXXCH motif close to the N terminus. The N-terminal two-thirds of cytochrome-c552 cover spatial regions around the heme prosthetic group that are similar to those observed for other cytochromes. The actual secondary structural elements that are responsible for that shielding do not, however, correlate well to other structures. Only the N-terminal helix (containing the heme binding cysteine residues) aligns reasonably well with other class I cytochromes. The most striking differences that distinguish the present structure from all other class I cytochromes is the C-terminal one-third of the molecule that wraps around the remainder of the structure as a stabilizing clamp, the existence of an extended beta-sheet covering one edge of the heme and the lack of any internal water molecule.

Dimer interface of glutathione S-transferase from Arabidopsis thaliana: influence of the G-site architecture on the dimer interface and implications for classification.

The three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana has been solved at 2.2 A resolution (Reinemer et al., 1996). The enzyme forms a dimer of two identical subunits. The structure shows a new G-site architecture and a novel and unique dimer interface. Each monomer of the protein forms a separate G-site. Therefore, the requirements on the dimer interface are reduced. As a consequence, the interactions between the monomers are weaker and residues at the dimer interface are more variable. Thus, the dimer interface looses its relevance for a classification of plant glutathione S-transferases and the formation of heterodimers becomes even more difficult to predict.

A laser microprobe mass analysis of brain aluminum and iron in dementia pugilistica: comparison with Alzheimer's disease.

We report a laser microprobe mass analysis of aluminum and iron content in the hippocampus and in the inferior temporal cortex in 2 cases of dementia pugilistica (DP), 4 cases of Alzheimer's disease (AD), and 3 controls. There was a predominant accumulation of Al and Fe within neurofibrillary tangles (NFT) in both DP and AD cases. High levels of Al and Fe were also detected in the nuclei of NFT-free and NFT-containing neurons, as well as in neuropil probe sites in these cases. In both regions, NFT contained substantially higher levels of Al and Fe in DP compared to AD cases. These findings suggest the existence of an association between the deposition of Al and Fe and NFT formation, and support the possibility of a global dysregulation of Al and Fe transport in DP and AD.

Altered distribution of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit GluR2(4) and the N-methyl-D-aspartate receptor subunit NMDAR1 in the hippocampus of patients with temporal lobe epilepsy.

In patients with therapy-refractory temporal lobe epilepsy (TLE), alterations of glutamate receptors have been proposed as a mechanism for enhanced excitability. Using commercially available monoclonal antibodies specific for the N-methyl-D-aspartate (NMDA) receptor subunit NMDAR1 and for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit GluR2(4), we have examined the distribution of these polypeptides in human hippocampal tissue that was surgically removed from patients with intractable TLE. Surgical specimens were classified according to the presence of Ammon's horn sclerosis (AHS) or a focal lesion in the temporal lobe. Cell counts and a densitometric analysis of the immunoreactivity patterns were carried out for all hippocampal subfields. NMDAR1 and GluR2(4) levels were markedly reduced in patients with AHS, primarily in those subfields with substantial neuronal cell loss (in particular CA1, CA4 and CA3), compared to those seen in patients with focal lesions and in control specimens obtained at autopsy. In contrast, the molecular layer of the dentate gyrus (DG-ML) showed significantly higher levels of GluR2(4) immunoreactivity in AHS compared to control tissue, while NMDAR1 showed no significant up-regulation in this sublayer. When the receptor staining intensity was normalized for alterations in neuronal density, no significant alterations could be detected except for an increase in GluR2(4) in the DG-ML of patients with AHS. These changes may reflect synaptic reorganization observed in the DG-ML of specimens from patients with chronic intractable TLE.

The 1.8 A crystal structure of human cathepsin G in complex with Suc-Val-Pro-PheP-(OPh)2: a Janus-faced proteinase with two opposite specificities.

The crystal structure of human neutrophil cathepsin G, complexed with the peptidyl phosphonate inhibitor Suc-Val-Pro-PheP-(OPh)2, has been determined to a resolution of 1.8 A using Patterson search techniques. The cathepsin G structure shows the polypeptide fold characteristic of trypsin-like serine proteinases and is especially similar to rat mast cell proteinase II. Unique to cathepsin G, however, is the presence of Glu226 (chymotrypsinogen numbering), which is situated at the bottom of the S1 specificity pocket, dividing it into two compartments. For this reason, the benzyl side chain of the inhibitor PheP residue does not fully occupy the pocket but is, instead, located at its entrance. Its positively charged equatorial edge is involved in a favourable electrostatic interaction with the negatively charged carboxylate group of Glu226. Arrangement of this Glu226 carboxylate would also allow accommodation of a Lys side chain in this S1 pocket, in agreement with the recently observed cathepsin G preference for Lys and Phe at P1. The cathepsin G complex with the covalently bound phosphonate inhibitor mimics a tetrahedral substrate intermediate. A comparison of the Arg surface distributions of cathepsin G, leukocyte elastase and rat mast cell protease II shows no simple common recognition pattern for a mannose-6-phosphate receptor-independent targeting mechanism for sorting of these granular proteinases.

A laser microprobe mass analysis of trace elements in brain mineralizations and capillaries in Fahr's disease.

We report a detailed analysis of the content of aluminum, iron, zinc, copper, calcium, and magnesium in the non-vascular and pericapillary mineralizations and the normal capillaries of the globus pallidus and dentate nucleus of the cerebellum in two patients with clinically and neuropathologically confirmed Fahr's disease. The study employed laser microprobe mass analysis, a technique that enables highly sensitive detection of the levels of trace elements. In the globus pallidus, there was a significant increase in aluminum-, iron-, zinc-, and calcium-related peak intensity in the pericapillary and non-vascular mineralizations compared to the normal capillaries. The pericapillary and non-vascular mineralizations had comparable concentrations of these elements. No difference was found in copper levels between the different probe sites. Magnesium was almost absent in pericapillary mineralizations and normal capillaries, while it accumulated within non-vascular mineralizations. In the cerebellar dentate nucleus, non-vascular mineralizations displayed higher concentrations of all of these elements than normal capillaries, while pericapillary mineralizations had a higher aluminum and lower iron, copper, and calcium content than did non-vascular mineralizations. Zinc and magnesium were selectively deposited within the non-vascular mineralizations in this nucleus. Furthermore, the element composition of non-vascular mineralizations differed between the globus pallidus and dentate nucleus. These findings indicate that the formation of pericapillary and non-vascular mineralizations may be two independent phenomena which coexist in the course of Fahr's disease. The marked qualitative and quantitative differences in trace element content in non-vascular mineralizations between the globus pallidus and cerebellar dentate nucleus suggest that the involvement of trace elements in the pathogenesis of Fahr's disease is probably indirect.

Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture.

Glutathione S-transferases (GST) are a family of multifunctional enzymes involved in the metabolization of a broad variety of xenobiotics and reactive endogenous compounds. The interest in plant glutathione S-transferases may be attributed to their agronomic value, since it has been demonstrated that glutathione conjugation for a variety of herbicides is the major resistance and selectivity factor in plants. The three-dimensional structure of glutathione S-transferase from the plant Arabidopsis thaliana has been solved by multiple isomorphous replacement and multiwavelength anomalous dispersion techniques at 3 A resolution and refined to a final crystallographic R-factor of 17.5% using data from 8 to 2.2 A resolution. The enzyme forms a dimer of two identical subunits each consisting of 211 residues. Each subunit is characterized by the GST-typical modular structure with two spatially distinct domains. Domain I consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side by an irregular segment containing three short 3(10)-helices, while domain II is entirely helical. The dimeric molecule is globular with a prominent large cavity formed between the two subunits. The active site is located in a cleft situated between domains I and II and each subunit binds two molecules of a competitive inhibitor S-hexylglutathione. Both hexyl moieties are oriented parallel and fill the H-subsite of the enzyme's active site. The glutathione peptide of one inhibitor, termed productive binding, occupies the G-subsite with multiple interactions similar to those observed for other glutathione S-transferases, while the glutathione backbone of the second inhibitor, termed unproductive binding, exhibits only weak interactions mediated by two polar contacts. A most striking difference from the mammalian glutathione S-transferases, which share a conserved catalytic tyrosine residue, is the lack of this tyrosine in the active site of the plant glutathione S-transferase.

Cellular distribution of the iron-binding protein lactotransferrin in the mesencephalon of Parkinson's disease cases.

Changes in the distribution of the iron-binding protein lactotransferrin have recently been described in the central nervous system during a variety of neurodegenerative disorders. To investigate whether lactotransferrin is associated with the neuropathological changes that characterize Parkinson's disease, we analyzed the distribution of this protein in the mesencephalon of neurologically normal individuals and patients affected with Parkinson's disease using quantitative immunohistochemical methods. High levels of lactotransferrin were observed in a large population of neurons in the substantia nigra of control cases. Lactotransferrin-positive neurons were severely affected by the neurodegenerative process that occurs in Parkinson's disease as indicated by a severe decrease in the number of immunolabeled neurons in all of these cases. Quantitative analysis also demonstrated higher immunolabeling levels of lactotransferrin in the surviving neurons in the substantia nigra and ventral tegmental area of Parkinson's disease cases compared to control cases. These results suggest that lactotransferrin may participate actively in the mechanism of neuronal degeneration in Parkinson's disease.

Immunocytochemical localization of non-NMDA ionotropic excitatory amino acid receptor subunits in human neocortex.

The distribution of immunocytochemically localized subunits that comprise ionotropic non-NMDA excitatory amino acid receptors was examined in human frontal, parietal and temporal association neocortex. AMPA/kainate receptor subunits were identified using a monoclonal antibody (3A11) that recognizes an epitope common to GluR2 and GluR4 [GluR2(4)], as well as polyclonal antisera that recognize GluR2 and GluR3 (GluR2/3). Kainate receptor subunits were identified using a monoclonal antibody (4F5) that recognizes an epitope common to GluR5/6/7. For all three antibodies used, labeling was observed in a large number of neurons throughout the human association neocortex with the highest immunoreactivity present in pyramidal-like neurons, a cellular pattern largely similar to that observed in the monkey neocortex. These data demonstrate the cellular localization patterns for some non-NMDA receptor subunits in human neocortex, details upon which further studies on the roles of these subunits in human neurological diseases can be based.