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Importance: Herpes simplex virus (HSV) hepatitis is a rare condition with a high mortality rate. Immunocompromised individuals, including pregnant women, are the most susceptible. When primary infection occurs during pregnancy, risk for disseminated HSV is greatly increased. Disseminated HSV can manifest in the form of HSV hepatitis., Objective: We aim to review the literature and summarize what is known about HSV hepatitis in pregnancy to aid in the diagnosis and treatment of this condition., Evidence Acquisition: A literature search of PubMed and Web of Science was performed. A total of 237 citations were found. All citations were independently reviewed. Thirty-eight full-text articles were identified and included in this review. Additional data from 1 unpublished case from our institution was included., Results: Fifty-six cases were included with average gestational age at diagnosis of 30 weeks. Patients presented with a wide variety of gastrointestinal, respiratory, neurologic, and urogenital symptoms. The most common examination findings were fever and abdominal tenderness. Only 18.2% of patients had a vesicular rash. All patients had a transaminitis, and 85% had positive viral cultures. A multitude of treatments were used with the majority of favorable outcomes occurring after treatment with acyclovir., Conclusions and Relevance: Although HSV hepatitis is rare, it carries a mortality rate of up to 39% for mothers and neonates. Therefore, it is crucial that HSV hepatitis be included on the differential diagnosis when a patient presents with fever and transaminitis. When HSV hepatitis is suspected, empiric therapy with acyclovir can be initiated with no additional risk to the fetus.

Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson's disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes.

Parkinson's disease is characterized by motor deficits caused by loss of midbrain dopaminergic neurons. Neurotrophic factors and cell transplantation have partially restored function in models of Parkinson's disease, but have had limited effects in humans. Here we show that intracerebroventricular administration of platelet-derived growth factor-BB can offer an alternative strategy to restore function in Parkinson's disease; In animal models of nigrostriatal injury, a two weeks treatment with platelet-derived growth factor-BB resulted in long-lasting restoration of striatal dopamine transporter binding sites and expression of nigral tyrosine hydroxylase. It also normalized amphetamine-induced rotational behavior in 6-hydroxydopamine lesioned rats. Platelet-derived growth factor-BB promoted proliferation of neural progenitor cells in the subventricular zone. The effects on dopaminergic neurons and functional recovery could be blocked by co-infusion with a proliferation inhibitor, indicating a link between the proliferative and anti-parkinsonian effects. Based on the current data, we consider platelet-derived growth factor-BB a clinical candidate drug for treatment of Parkinson's disease.

The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

Alpha-synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.

Pathologic accumulation of alpha-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of alpha-synuclein in vivo was indicated by the detection of alpha-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal alpha-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of alpha-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with alpha-synuclein within nigral dopaminergic neurons of paraquat-treated and alpha-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of alpha-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of alpha-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal alpha-synuclein buildup.

Because of its normal function in synaptic plasticity and pathologic involvement in age-related neurodegenerative diseases, the protein alpha-synuclein could play an important role in aging processes. Here we compared alpha-synuclein expression in the substantia nigra and other brain regions of young (2-month-old), middle-aged (10-month-old), and old (20-month-old) mice. Levels of nigral alpha-synuclein mRNA, as assessed by both in situ hybridization and qPCR, were high in young mice and progressively declined in middle-aged and old animals. This age-dependent mRNA loss was paralleled by a marked reduction of alpha-synuclein protein; immunoreactivity of midbrain sections stained with an anti-alpha-synuclein antibody was most robust in 2-month-old mice and weakest in 20-month-old animals. Lowering of nigral alpha-synuclein could not be explained by a loss of dopaminergic neurons and was relatively specific since no change in beta-synuclein mRNA and protein occurred with advancing age. Finally, age-related decreases in alpha-synuclein were widespread throughout the mouse brain, affecting other regions (e.g., hippocampus) besides the substantia nigra. The data suggest that loss of alpha-synuclein could contribute to or be a marker of synaptic dysfunction in the aging brain. They also emphasize important differences in alpha-synuclein expression between rodents and primates since earlier reports have shown a marked elevation of alpha-synuclein protein in the substantia nigra of older humans and non-human primates.

alpha-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic accumulation of alpha-synuclein and protein modifications (i.e. nitration, phosphorylation, and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of alpha-synuclein but not of beta-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy also revealed large numbers of dystrophic axons labeled with alpha-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) alpha-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. After toxicant exposure, alpha-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for alpha-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce alpha-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of alpha-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.

Paraquat (PQ) causes selective degeneration of dopaminergic neurons in the substantia nigra pars compacta, reproducing an important pathological feature of Parkinson disease. Oxidative stress, c-Jun N-terminal kinase activation, and alpha-synuclein aggregation are each induced by PQ, but details of the cell death mechanisms involved remain unclear. We have identified a Bak-dependent cell death mechanism that is required for PQ-induced neurotoxicity. PQ induced morphological and biochemical features that were consistent with apoptosis, including dose-dependent cytochrome c release, with subsequent caspase-3 and poly(ADP-ribose) polymerase cleavage. Changes in nuclear morphology and loss of viability were blocked by cycloheximide, caspase inhibitor, and Bcl-2 overexpression. Evaluation of Bcl-2 family members showed that PQ induced high levels of Bak, Bid, BNip3, and Noxa. Small interfering RNA-mediated knockdown of BNip3, Noxa, and Bak each protected cells from PQ, but Bax knockdown did not. Finally, we tested the sensitivity of Bak-deficient mice and found them to be resistant to PQ treatments that depleted tyrosine hydroxylase immuno-positive neurons in the substantia nigra pars compacta of wild-type mice.

Mutations in the gene for DJ-1 have been associated with early-onset autosomal recessive parkinsonism. Previous studies of null DJ-1 mice have shown alterations in striatal dopamine (DA) transmission with no DAergic cell loss. Here we characterize a new line of DJ-1-deficient mice. A subtle locomotor deficit was present in the absence of a change in striatal DA levels. However, increased [(3)H]-DA synaptosomal uptake and [(125)I]-RTI-121 binding were measured in null DJ-1 vs. wild-type mice. Western analyses of synaptosomes revealed significantly higher dopamine transporter (DAT) levels in pre-synaptic membrane fractions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure exacerbated striatal DA depletion in null DJ-1 mice with no difference in DAergic nigral cell loss. Furthermore, increased 1-methyl-4-phenylpyridinium (MPP(+)) synaptosomal uptake and enhanced MPP(+) accumulation were measured in DJ-1-deficient vs. control striatum. Thus, under null DJ-1 conditions, DAT changes likely contribute to altered DA neurotransmission and enhanced sensitivity to toxins that utilize DAT for nigrostriatal entry.

The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is responsible for packaging dopamine into vesicles for subsequent release and has been suggested to serve a neuroprotective role in the dopamine system. Here, we show that mice that express approximately 5% of normal VMAT2 (VMAT2 LO) display age-associated nigrostriatal dopamine dysfunction that ultimately results in neurodegeneration. Elevated cysteinyl adducts to L-DOPA and DOPAC are seen early and are followed by increased striatal protein carbonyl and 3-nitrotyrosine formation. These changes were associated with decreased striatal dopamine and decreased expression of the dopamine transporter and tyrosine hydroxylase. Furthermore, we observed an increase in alpha-synuclein immunoreactivity and accumulation and neurodegeneration in the substantia nigra pars compacta in aged VMAT2 LO mice. Thus, VMAT2 LO animals display nigrostriatal degeneration that begins in the terminal fields and progresses to eventual loss of the cell bodies, alpha-synuclein accumulation, and an L-DOPA responsive behavioral deficit, replicating many of the key aspects of Parkinson's disease. These data suggest that mishandling of dopamine via reduced VMAT2 expression is, in and of itself, sufficient to cause dopamine-mediated toxicity and neurodegeneration in the nigrostriatal dopamine system. In addition, the altered dopamine homeostasis resulting from reduced VMAT2 function may be conducive to pathogenic mechanisms induced by genetic or environmental factors thought to be involved in Parkinson's disease.

Numerous epidemiological studies have shown an association between pesticide exposure and an increased risk of developing Parkinson's disease (PD). Here, we provide evidence that the insecticide dieldrin causes specific oxidative damage in the nigrostriatal dopamine (DA) system. We report that exposure of mice to low levels of dieldrin for 30 days resulted in alterations in dopamine-handling as evidenced by a decrease in dopamine metabolites, DOPAC (31.7% decrease) and HVA (29.2% decrease) and significantly increased cysteinyl-catechol levels in the striatum. Furthermore, dieldrin resulted in a 53% decrease in total glutathione, an increase in the redox potential of glutathione, and a 90% increase in protein carbonyls. Alpha-synuclein protein expression was also significantly increased in the striatum (25% increase). Finally, dieldrin caused a significant decrease in striatal expression of the dopamine transporter as measured by (3)H-WIN 35,428 binding and (3)H-dopamine uptake. These alterations occurred in the absence of dopamine neuron loss in the substantia nigra pars compacta. These effects represent the ability of low doses of dieldrin to increase the vulnerability of nigrostriatal dopamine neurons by inducing oxidative stress and suggest that pesticide exposure may act as a promoter of PD.

Dopaminergic cells in the substantia nigra are highly vulnerable to the neurodegenerative process of Parkinson's disease. Therefore, mechanisms that enhance their susceptibility to injury bear important implications for disease pathogenesis. Repeated injections with the herbicide paraquat cause oxidative stress and a selective loss of dopaminergic neurons in mice. In this model, the first paraquat exposure, though not sufficient to induce any neurodegeneration, predisposes neurons to damage by subsequent insults. The purpose of this study was to elucidate the mechanisms underlying this "priming" event. We found that a single paraquat exposure was followed by an increase in the number of cells with immunohistochemical, morphological and biochemical characteristics of activated microglia, including induction of NADPH oxidase. If this microglial response was inhibited by the anti-inflammatory drug minocycline, subsequent exposures to the herbicide failed to cause oxidative stress and neurodegeneration. On the other hand, if microglial activation was induced by pre-treatment with lipopolysaccharide, a single paraquat exposure became capable of triggering a loss of dopaminergic neurons. Finally, mutant mice lacking functional NADPH oxidase were spared from neurodegeneration caused by repeated paraquat exposures. Data indicate that microglial activation and consequent induction of NADPH oxidase may act as risk factors for Parkinson's disease by increasing the vulnerability of dopaminergic cells to toxic injury.

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.

The vulnerability of different dopaminergic cell populations to damage caused by the herbicide paraquat was assessed by stereological counts of tyrosine hydroxylase-positive and calbindin-D28k-immunoreactive neurons in A9 (substantia nigra pars compacta) and A10 (ventral tegmental area and other cell groups). In saline-treated control mice, tyrosine hydroxylase-immunoreactive neurons represented 80% and 45% of the total neuronal population in A9 and A10, respectively, and the number of calbindin-D28k-positive neurons was five times greater in A10 than A9. Sequential injections with paraquat resulted in a significant loss of dopaminergic neurons in A9. In contrast, tyrosine hydroxylase-positive cells in A10 were spared from paraquat-induced degeneration. Furthermore, expression of calbindin-D28k was consistently associated with neuronal resistance to the herbicide in both A9 and A10. Paraquat exposure also induced oxidative stress as indicated by an increase in the number of midbrain cells positive for 4-hydroxy-2-nonenal, a marker of lipid peroxidation. Co-localization studies revealed that calbindin-D28k immunoreactivity overlapped with tyrosine hydroxylase labeling and that, after paraquat administration, (i) the vast majority of midbrain 4-hydroxy-2-nonenal-immunoreactive cells were dopaminergic (tyrosine hydroxylase-immunoreactive), (ii) tyrosine hydroxylase/4-hydroxy-2-nonenal-positive neurons were much more prevalent in A9 than A10, and (iii) all calbindin-D28k-containing neurons were characterized by lack of lipid peroxidation (4-hydroxy-2-nonenal immunoreactivity). Results in this paraquat model emphasize that, despite sharing a similar dopaminergic phenotype, different groups of midbrain neurons vary dramatically in their vulnerability to injury. Data also indicate that these differences are attributable, at least in part, to a varying susceptibility of dopaminergic cell populations to oxidative stress.

Mammalian puberty requires activation of luteinizing hormone-releasing hormone (LHRH) neurons. In turn, these neurons are controlled by transsynaptic and glia-to-neuron communication pathways, which employ diverse cellular proteins for proper function. We have now used a high throughput relative quantitative proteomics technique to identify such proteins. We selected the method of two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) and cleavable isotope-coded affinity tags (cICAT), to both identify and quantify individual proteins within a complex protein mixture. The proteins used derived from the hypothalamus of juvenile (25-day-old) and peripubertal (first proestrus, LP) female rats, and their identity was established by analyzing their mass spectra via database searching. Five proteins involved in glutamate metabolism were detected and two of them appeared to be differentially expressed. They were selected for further analysis, because of their importance in controlling glutamate synthesis and degradation, and their preferential expression in astroglial cells. One, glutamate dehydrogenase (GDH) catalyzes glutamate synthesis; its hypothalamic content detected by 2DLC-MS/MS increases at first proestrus. The other, glutamine synthetase (GS), catalyzes the metabolism of glutamate to glutamine; its content decreases in proestrus. Western blot analysis verified these results. Because these changes suggested an increased glutamate production at puberty, we measured glutamate release from hypothalamic fragments from juvenile 29-day old rats, and from rats treated with PMSG to induce a premature proestrus surge of luteinizing hormone (LH). To determine the net output of glutamate in the absence of re-uptake we used the excitatory amino acid transporter (EAAT) inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). PDC elicited significantly more glutamate- and LHRH-release from the proestrus hypothalamus. Thus, an increase excitatory drive to the LHRH neuronal network provided by glutamatergic inputs of glial origin, is an event contributing to the pubertal activation of LHRH secretion.

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.

Changes in the expression of alpha-synuclein are likely to underlie its normal function as well as its role in pathological processes. The relationship between toxic injury and alpha-synuclein expression was assessed in the substantia nigra of squirrel monkeys treated with a single injection of MPTP and sacrificed 1 week or 1 month later. At 1 week, when stereological cell counting revealed only a small decrease (-10%) in the number of dopaminergic neurons, alpha-synuclein mRNA and protein were markedly enhanced. Increased alpha-synuclein immunoreactivity was evident at the level of neuronal fibers whereas nigral cell bodies were devoid of detectable protein. At 1 month post-MPTP, neuronal loss rose to 40%. Both alpha-synuclein mRNA and protein remained elevated but, noticeably, a robust alpha-synuclein immunoreactivity characterized a significant number of cell bodies. Neuromelanin granules are hallmarks of dopaminergic neurons in primates. Therefore, the number of alpha-synuclein-positive cells that also contained neuromelanin was counted throughout the substantia nigra. At 1 month, the vast majority of alpha-synuclein-immunoreactive neurons contained neuromelanin, and approximately 80% of the dopaminergic cell bodies that survived MPTP toxicity stained positive for alpha-synuclein. The results indicate that a single toxic insult is capable of inducing a sustained alpha-synuclein up-regulation in the primate brain. They support a direct relationship between neuronal injury and enhanced alpha-synuclein expression, and suggest that protein elevation within cell bodies may be a late feature of neurons that have endured a toxic stress.

Systemic treatment of mice with the herbicide paraquat causes the selective loss of nigrostriatal dopaminergic neurons, reproducing the primary neurodegenerative feature of Parkinson's disease. To elucidate the role of oxidative damage in paraquat neurotoxicity, the time-course of neurodegeneration was correlated to changes in 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation marker. When mice were exposed to three weekly injections of paraquat, no nigral dopaminergic cell loss was observed after the first administration, whereas a significant reduction of neurons followed the second exposure. Changes in the number of nigral 4-HNE-positive neurons suggest a relationship between lipid peroxidation and neuronal death, since a dramatic increase in this number coincided with the onset and development of neurodegeneration after the second toxicant injection. Interestingly, the third paraquat administration did not cause any increase in 4-HNE-immunoreactive cells, nor did it produce any additional dopaminergic cell loss. Further evidence of paraquat-induced oxidative injury derives from the observation of nitrotyrosine immunoreactivity in the substantia nigra of paraquat-treated animals and from experiments with ferritin transgenic mice. These mice, which are characterized by a decreased susceptibility to oxidative stress, were completely resistant to the increase in 4-HNE-positive neurons and the cell death caused by paraquat. Thus, paraquat exposure yields a model that emphasizes the susceptibility of dopaminergic neurons to oxidative damage.

Oligosaccharyltransferase (OST) catalyzes the cotranslational transfer of high-mannose sugars to nascent polypeptides during N-linked glycosylation in the rough endoplasmic reticulum lumen. Nine OST subunits have been identified in yeast. However, the composition and organization of mammalian OST remain unclear. Using two-dimensional Blue Native polyacrylamide gel electrophoresis/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, we now demonstrate that mammalian OST can be isolated from solubilized, actively engaged ribosomes as multiple distinct protein complexes that range in size from approximately 500 to 700 kDa. These complexes exhibit different ribosome affinities and subunit compositions. The major complex, OSTC(I), had an apparent size of approximately 500 kDa and was readily released from ribosome translocon complexes after puromycin treatment under physiological salt conditions. Two additional complexes were released only after treatment with high salt: OSTC(II) ( approximately 600 kDa) and OSTC(III) ( approximately 700 kDa). Both remained stably associated with heterotrimeric Sec61alphabetagamma, while OSTC(III) also contained the tetrameric TRAP complex. All known mammalian OST subunits (STT3-A, ribophorin I, ribophorin II, OST48, and DAD1) were present in all complexes. In addition, two previously uncharacterized proteins were also copurified with OST. Mass spectrometry identified a 17 kDa protein as DC2 which is weakly homologous to the C-terminal half of yeast Ost3p and Ost6p. The second protein (14 kDa) was tentatively identified as keratinocyte-associated protein 2 (KCP2) and has no previously known function. Our results identify two potential new subunits of mammalian OST and demonstrate a remarkable heterogeneity in OST composition that may reflect a means for controlling nascent chain glycosylation.

With the increasing availability of de novo sequencing algorithms for interpreting high-mass accuracy tandem mass spectrometry (MS/MS) data, there is a growing need for programs that accurately identify proteins from de novo sequencing results. De novo sequences derived from tandem mass spectra of peptides often contain ambiguous regions where the exact amino acid order cannot be determined. One problem this poses for sequence alignment algorithms is the difficulty in distinguishing discrepancies due to de novo sequencing errors from actual genomic sequence variation and posttranslational modifications. We present a novel, mass-based approach to sequence alignment, implemented as a program called OpenSea, to resolve these problems. In this approach, de novo and database sequences are interpreted as masses of residues, and the masses, rather than the amino acid codes, are compared. To provide further flexibility, the masses can be aligned in groups, which can resolve many de novo sequencing errors. The performance of OpenSea was tested with three types of data: a mixture of known proteins, a mixture of unknown proteins that commonly contain sequence variations, and a mixture of posttranslationally modified known proteins. In all three cases, we demonstrate that OpenSea can identify more peptides and proteins than commonly used database-searching programs (SEQUEST and ProteinLynx) while accurately locating sequence variation sites and unanticipated posttranslational modifications in a high-throughput environment.

Increasing incidence of Parkinson's disease with advancing age suggests that age-related processes predispose the nigrostriatal dopaminergic system to neurodegeneration. Several hypotheses concerning the effects of aging on nigrostriatal neurons were assessed in this study using a non-human primate model. First, we examined the possibility that the total number of dopaminergic neurons decline in the substantia nigra as a function of age. Stereological counting based on both tyrosine hydroxylase immunoreactivity (TH-ir) and neuromelanin (NM) content revealed no difference in cell number between young, middle-aged and old squirrel monkeys. We then determined whether advancing age changed the relative proportion of neurons characterized by 1) TH-ir in the absence of NM, 2) the presence of both TH-ir and NM, or 3) NM without TH-ir. Indeed, a progressive age-related depletion of TH only cells was paralleled by an increase in NM only neurons. The possibility that these changes could underlie a functional impairment of the nigrostriatal system was supported by striatal dopamine measurements showing a decrease in older monkeys. Finally, we tested the hypotheses that aging may enhance cell vulnerability to injury and that different dopaminergic subpopulations display varying degrees of susceptibility. When monkeys were exposed to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, cell loss was markedly more pronounced in older animals, and the ranking of vulnerability was TH only < TH/NM < NM only cells. The data indicate that, even in the absence of an overall neuronal loss, changes in the characteristics of dopaminergic cells reflect functional deficits and increased vulnerability to injury with age. NM content appears to be an important marker of these age-related effects., (Copyright 2004 Wiley-Liss, Inc.)

Purpose: The purpose of this study is to compare the protein composition of the B-3 line of transformed human lens epithelial (HLE) cells to that of freshly dissected HLE cells. This provides baseline data on lens cell proteins from fresh lens cells and from the B-3 cell line, which is often used as a model system for the lens., Methods: Human lens epithelial cells adherent to the lens capsule were dissected into central (undifferentiated) and peripheral (partially differentiated) populations. Fully differentiated human lens fiber cells were isolated from the outer cortical layers of the lens. HLE B-3 cells were analyzed at several passage levels. Extracts were prepared from each cell type and the proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). Representative gel patterns were visually compared, spots excised, and trypsin digests prepared. The peptide compositions of the digests were analyzed using either liquid chromatography electrospray ionization tandem mass spectrometry or atmospheric pressure-matrix-assisted laser desorption ionization mass spectrometry, using a liquid chromatography classic ion trap (LCQ) mass spectrometer., Results: Two-DE patterns were obtained for fresh and cultured cell types. Similar patterns were observed between central and peripheral HLE cells, both of which contained high levels of alphaA-, alphaB-, and betaB2-crystallins; alpha-enolase; and aldehyde dehydrogenase. HLE B-3 cultured cells were characterized by a marked loss of crystallins and a relatively higher level of noncrystallin proteins--most notably, high molecular weight, acidic proteins. Whereas subunit d of adenosine triphosphate (ATP) synthase, alphaB-crystallin, galectin, glyceraldehyde-3-phosphate dehydrogenase, alpha-enolase, actin, peptidylprolyl isomerase A, phosphatidylethanolamine-binding protein, and vimentin were present in both fresh and cultured lens epithelium, only the high abundance of alpha-enolase, galectin-1, and vimentin suggested that B-3 cells were lens derived., Conclusions: Freshly dissected noncultured HLE cells from both central and peripheral regions contain a high concentration of crystallins that mask the detection of less abundant proteins by 2-DE. Transformation and culture of HLE cells causes a loss of these crystallins and an increase in the relative concentration of other proteins. However, most of these noncrystallin proteins were different from those observed in noncultured HLE cells. These results suggest that transformation markedly alters the protein expression pattern in immortalized HLE cells and that caution should be exercised when using them to study properties of HLE cells in vivo.

The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.

Alpha-synuclein is likely to play a role in neurodegenerative processes, including the degeneration of nigrostriatal dopaminergic neurons that underlies Parkinson's disease. However, the toxicological properties of alpha-synuclein remain relatively unknown. Here, the relationship between alpha-synuclein expression and neuronal injury was studied in mice exposed to the herbicide paraquat. Paraquat neurotoxicity was compared in control animals versus mice with transgenic expression of human alpha-synuclein driven by the tyrosine hydroxylase (TH) promoter. In control mice, paraquat caused both the formation of alpha-synuclein-containing intraneuronal deposits and the degeneration of nigrostriatal neurons, as demonstrated by silver staining and a reduction of the counts of TH-positive and Nissl-stained cells. Mice overexpressing alpha-synuclein, either the human wild-type or the Ala53Thr mutant form of the protein, displayed paraquat-induced protein aggregates but were completely protected against neurodegeneration. These resistant animals were also characterized by increased levels of HSP70, a chaperone protein that has been shown to counteract paraquat toxicity in other experimental models and could therefore contribute to neuroprotection in alpha-synuclein transgenic mice. The results indicate a dissociation between toxicant-induced alpha-synuclein deposition and neurodegeneration. They support a role of alpha-synuclein against toxic insults and suggest that its involvement in human neurodegenerative processes may arise not only from a gain of toxic function, as previously proposed, but also from a loss of defensive properties.

Exposure to the herbicide paraquat causes selective nigrostriatal degeneration and aggregation of alpha-synuclein in the mouse brain. The purpose of this study was to assess mechanisms of paraquat entry into the CNS and, in particular, the effects of substrates of the blood-brain barrier (BBB) neutral amino acid transporter (System L carrier) on paraquat accumulation and neurotoxicity. Using a paraquat antibody, robust immunoreactivity was observed in the midbrain of mice injected with the herbicide. This immunoreactivity was abolished by administration of l-valine or l-phenylalanine, two System L substrates, immediately before paraquat exposure. Pre-treatment with these amino acids completely protected against paraquat-induced loss of nigrostriatal dopaminergic cells and formation of thioflavine S-positive intracellular deposits. Interestingly, the anti-parkinsonian drug l-dopa, which is transported across the BBB through the same neutral amino acid carrier, was also neuroprotective when administered 30 min prior to paraquat. In contrast, paraquat-induced toxicity was unaffected if animals (i) were pre-treated with d-valine, the biologically inactive d-isomer of l-valine, or with l-lysine, a substrate of the basic rather than the neutral amino acid carrier, or (ii) were injected with l-dopa 24 h after paraquat exposure. Data are consistent with a critical role of uptake across the BBB in paraquat neurotoxicity, and suggest that dietary elements (e.g. amino acids) or therapeutic agents (e.g. l-dopa) may modify the effects of toxicants targeting the nigrostriatal system.

One of the most useful treatments of Parkinson's disease (PD) is dihydroxyphenylalanine (L-DOPA) administration. However, L-DOPA has been suggested to be toxic to dopamine (DA) neurons and perhaps contribute to the progression of the disease. Sequestration of DA and dopaminergic neurotoxins into vesicles by the vesicular monoamine transporter 2 (VMAT2) is a key factor in preventing cellular damage. Mice with reduced expression of VMAT2 (VMAT2 heterozygote knockout mice; VMAT2 (+/-)) are more sensitive to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine. In this study, we subjected VMAT2 (+/-) mice to subchronic administration of L-DOPA to determine if it was toxic in this model. VMAT2 wild-type (VMAT2 (+/+)) and VMAT2 (+/-) mice were given i.p. injections of L-DOPA:carbidopa (50:5 mg/kg) three times a day for 28 days. Biochemical analysis revealed a significant increase in striatal DA levels in both groups of mice treated with L-DOPA. L-DOPA treatment significantly decreased DAT levels in VMAT2 (+/+) mice, but not in VMAT2 (+/-) mice. VMAT2 protein levels, an index of terminal integrity and the number of tyrosine hydroxylase (TH)-positive nigral cells remained unchanged after L-DOPA treatment. These data indicate that in an animal model that displays increased susceptibility to dopaminergic injury, a subchronic administration of L-DOPA does not induce toxicity.

Buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, induces oxidative cataracts following multiple injections into mice at 1 week of age. Cultures of lenses with (35)S-methionine have previously demonstrated altered patterns of protein biosynthesis that precede and accompany these cataracts. To obtain parallel information about changes in protein phosphorylation during cataract development, lenses from BSO-treated or control mouse pups were cultured for 3 hr at 37 degrees C with (32)P(i), homogenized in phosphate buffer, and resolved by centrifugation into water-soluble (WS) and water-insoluble (WI) fractions. These were characterized by 2D-gel electrophoresis, Coomassie blue staining, phosphorimaging, immunoblotting, and tandem mass spectrometry. Heaviest labelling was in the WI fraction. The labelled 2D-gel spots included: (1) a series of phosphorylated filensins at 95 kDa; (2) a major radioactive spot at 45-50 kDa, slightly anodic to actin and the beaded filament protein, phakinin (CP 49); (3) a phosphorylated betaB1-crystallin, considerably anodic to parent betaB1; (4) an acidic cluster of labelled alphaA-crystallins, phosphorylated in part at serine-148, and (5) a labelled trace alpha crystallin, slightly anodic to alphaB-crystallin. The results confirm previously reported phosphorylations of actin, phakinin, alphaA- and alphaB-crystallin, demonstrate previously unrecognized phosphorylations of filensin and betaB1-crystallin, and provide unequivocal evidence for phosphorylation of alphaA-crystallin at serine-148. The earliest changes in phosphorylation detected after BSO treatment were increased labelling of alphaA- and alphaB-crystallin during cataract stages 1-3, coupled with a general decrease in protein labelling. In stage 5 cataracts, phosphorylated alpha crystallins persisted as the dominant labelled species. However, the major modifications of alphaA-crystallin in advanced BSO cataracts were unlabelled and partially degraded, in contrast to phosphorylated alphaA. It is therefore proposed that phosphorylation of alphaA-crystallin may confer resistance to proteolytic degradation.

Environmental toxicants and, in particular, pesticides have been implicated as risk factors in Parkinson's disease (PD). The purpose of this study was to determine if selective nigrostriatal degeneration could be reproduced by systemic exposure of mice to the widely used herbicide paraquat. Repeated intraperitoneal paraquat injections killed dopaminergic neurons in the substantia nigra (SN) pars compacta, as assessed by stereological counting of tyrosine hydroxylase (TH)-immunoreactive and Nissl-stained neurons. This cell loss was dose- and age-dependent. Several lines of evidence indicated selective vulnerability of dopaminergic neurons to paraquat. The number of GABAergic cells was not decreased in the SN pars reticulata, and counting of Nissl-stained neurons in the hippocampus did not reveal any change in paraquat-treated mice. Degenerating cell bodies were observed by silver staining, but only in the SN pars compacta, and glial response was present in the ventral mesencephalon but not in the frontal cortex and cerebellum. No significant depletion of striatal dopamine followed paraquat administration. On the other hand, enhanced dopamine synthesis was suggested by an increase in TH activity. These findings unequivocally show that selective dopaminergic degeneration, one of the pathological hallmarks of PD, is also a characteristic of paraquat neurotoxicity. The apparent discrepancy between pathological (i.e., neurodegeneration) and neurochemical (i.e., lack of significant dopamine loss) effects represents another important feature of this paraquat model and is probably a reflection of compensatory mechanisms by which neurons that survive damage are capable of restoring neurotransmitter tissue levels.

alpha-Synuclein-containing aggregates represent a feature of a variety of neurodegenerative disorders, including Parkinson's disease (PD). However, mechanisms that promote intraneuronal alpha-synuclein assembly remain poorly understood. Because pesticides, particularly the herbicide paraquat, have been suggested to play a role as PD risk factors, the hypothesis that interactions between alpha-synuclein and these environmental agents may contribute to aggregate formation was tested in this study. Paraquat markedly accelerated the in vitro rate of alpha-synuclein fibril formation in a dose-dependent fashion. When mice were exposed to the herbicide, brain levels of alpha-synuclein were significantly increased. This up-regulation followed a consistent pattern, with higher alpha-synuclein at 2 days after each of three weekly paraquat injections and with protein levels returning to control values by day 7 post-treatment. Paraquat exposure was also accompanied by aggregate formation. Thioflavine S-positive structures accumulated within neurons of the substantia nigra pars compacta, and dual labeling and confocal imaging confirmed that these aggregates contained alpha-synuclein. The results suggest that up-regulation of alpha-synuclein as a consequence of toxicant insult and direct interactions between the protein and environmental agents are potential mechanisms leading to alpha-synuclein pathology in neurodegenerative disorders.

Ubiquitous type m-calpain and lens specific Lp82 calpain were separated and partially purified from fetal bovine lens and the enzymatic characteristics were compared. Lens m-calpain required 200 microM calcium for 1/2 maximal activity, while Lp82 required 30 microM. Both types of calpains were inhibited by 0.1 mM E64, and 5 mM iodoacetamide, but not by 1 mM phenylmethylsulfonyl fluoride. Lp82 was insensitive to 1 microM calpastatin peptide while m-calpain was effectively inhibited. In the presence of calcium, m-calpain lost most of its activity within 2 hr, while Lp82 was continually active for 18 hr. Both calpains cleaved the natural substrates betaA3 and alphaB crystallins in a similar manner. However, incubation of alphaA crystallin with m-calpain removed ten amino acid residues from its C-terminus, while incubation with Lp82 removed only five residues. The latter truncation product of alphaA was also found in vivo. These data suggested that Lp82 may have a more important role than m-calpain in modification of crystallins during lens maturation., ((C) 2001 Academic Press.)

We report a strategy for high through-put sequence analyses of large MHC class II-bound peptide repertoires which combines automated electrospray ionization tandem mass-spectrometry with computer-assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I-Ab in activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H-2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II-bound self peptides derived from cytosolic proteins exists in vivo.

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.

The beta subunits of voltage-dependent calcium channels (VDCC) modulate the electrophysiology and cell surface expression of pore-forming alpha1 subunits. In the present study we have investigated the distribution of beta1,beta2,beta3 and beta4 in the human hippocampus using in situ hybridization (ISH) and immunohistochemistry. ISH studies showed a similar distribution of expression of beta1,beta2 and beta3 subunit mRNAs, including labelling of the dentate granule cell layer, all CA pyramidal regions, and the subiculum. Relatively low levels of expression of beta1 and beta2 subunit mRNAs correlated with low protein expression in the immunocytochemical (ICC) studies. There was a relative lack of beta4 expression by both ISH and ICC in the CA1 region, compared with high levels of expression in the subiculum. Immunostaining for beta1 and beta2 subunits was weak and relatively homogeneous throughout the hippocampus. The beta3 and beta4 subunits appeared to be more discretely localized. In general, beta3-immunoreactivity was moderate both in cell bodies, and as diffuse staining in the surrounding neuropil. Strongest staining was observed in mossy fibres and their terminal region in the CA3 stratum lucidum. In contrast, beta4-immunoreactivity in the neuropil showed intense dendritic localisation. Unlike the other subunits, beta4-immunoreactivity was absent from CA1 pyramidal neurones but was present in a small population of interneurone-like cells. The localisation of beta3 and beta4 may represent presynaptic and postsynaptic compartments in some populations of hippocampal neurones. Comparison of beta subunit distribution with previously published data on alpha1 subunits indicates certain neuronal groups and subcellular compartments in which the subunit composition of native pre- and postsynaptic VDCC can be predicted., (Copyright 1998 Elsevier Science B.V.)

The alpha(1) subunit provides both the voltage-sensing mechanism and the ion pore of voltage-dependent calcium channels. Of the six classes of alpha(1) subunit cloned to date, alpha)1A) is the subject of debate in terms of its functional correlate, although it is generally thought to encode voltage-dependent calcium channels of the omega-agatoxin IVA-sensitive, P/Q type. In the present study, an alpha(1A)-specific riboprobe and antibody were used with in situ hybridisation and immunohistochemical techniques to localise alpha(1A) messenger ribonucleic acid transcripts and subunit protein throughout the mature rat brain. Dual localisation of alpha(1A) protein and markers for acetylcholine, catecholamines, and 5-hydroxytryptamine have also been performed in a number of discrete areas. Abundant and widespread distribution of alpha(1A) protein was found, with immunoreactivity occurring both in cell bodies and as punctate staining in areas of neuronal processes. Several associations were noted across alpha(1A) localisation, defined neuroanatomical regions, and neurotransmitter systems. However, alpha(1A) expression was not confined to loci corresponding to any one neurotransmitter type, although a high level of expression was observed in cholinergic neurones. The distribution of the alpha(1A) subunit in the rat corresponded well with the limited human mapping data that are available.

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits.

The molecular, structural and functional characterisation of ion channels in the CNS forms an area of intense investigation in current brain research. For strategic and logistical reasons, rodents have historically been the species of choice for these studies. The examination of human CNS tissues generally presents the investigator with specific challenges that are often less problematic in animal studies, e.g. post-mortem delay/agonal status, and thus both the experimental design and techniques must be manipulated accordingly. Since much pharmaceutical interest is currently focused on neuronal ion channels, the examination of their expression in human brain material is of particular importance. We describe here the details of methods that we have developed and used successfully in the study of the expression of voltage-dependent calcium channels (VDCCs) in human CNS tissues. Presynaptic neuronal VDCCs control neurotransmitter release and are important new drug targets. They are composed of three subunits, alpha 1, beta and alpha 2/delta and multiple gene classes of each protein have been identified. Little is known, however, about the distribution of neuronal VDCCs in the human central nervous system, although initial studies have been performed in rat and rabbit.

Methods to identify proteins contained in mixtures are described. The approach uses microcolumn liquid chromatography and automated tandem mass spectrometry in conjunction with protein and nucleotide database searching algorithms. This approach is applied to the identification of proteins obtained by immunoprecipitation reactions, interaction with a GST protein fusion products and interaction with a macromolecular complex.

A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of proteins is digested with a proteolytic enzyme to produce a large collection of peptides. The complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra. The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from standard protein mixtures show that proteins present in simple mixtures can be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within the mixture can be identified at low femtomole levels. Based on these studies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interaction with a protein-protein fusion product, and specific interaction with a macromolecular complex. The approach described in this article provides a rapid method for the direct identification of proteins in mixtures.

Understanding the structural features of naturally processed peptides found within the major histocompatibility complex (MHC) class II peptide binding groove from disease-associated MHC molecules may provide insights into the nature of potential disease-related antigens. Class II MHC/peptide complexes were purified by immunoaffinity from transformed B cell lines homozygous for DRB1*0404 (an allele associated with rheumatoid arthritis) and *0402 (a closely related allele not associated with this disease). Peptides were eluted at acidic pH, fractionated by reversed phase HPLC, and analyzed by capillary electrophoresis. Those fractions containing a single dominant peptide were sequenced by automated Edman degradation and tandem mass spectrometry. The predominant peptide species identified came from non-polymorphic regions of the HLA class I molecules expressed by each cell line. Peptides from DRB1*0404 were found to be nested clusters derived from positions 26-43 of the HLA-B and -C alpha-chain. DRB1*0402 contained as the predominant peptide species a nested cluster from positions 129-145 of the HLA-B alpha-chain. The primary structure of the class I derived peptides was consistent with that seen by peptides exhibiting promiscuous DR binding behavior. Processing of MHC-derived peptides by MHC class II molecules is a common occurrence in the transformed B cell lines analyzed.

An overview is provided of methods for the study of complex biological processes by using micro-column liquid chromatography-electrospray ionization tandem mass spectrometry. Procedures discussed include electrospray ionization, micro-column liquid chromatography, tandem mass spectrometry, tandem mass spectra data interpretation for peptides, and database searching with mass spectral data. Several problems in immunology are discussed to illustrate this approach.