Alpha-synuclein is a major component of Lewy bodies and glial cytoplasmic inclusions, pathological hallmarks of idiopathic Parkinson's disease and multiple system atrophy, and it is assumed to be aetiologically involved in these conditions. However, the quantitative status of brain alpha-synuclein in different Parkinsonian disorders is still unresolved and it is uncertain whether alpha-synuclein accumulation is restricted to regions of pathology. We compared membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein, both the full-length 17 kDa and high molecular weight species, by western blotting in autopsied brain of patients with Parkinson's disease (brainstem-predominant Lewy body disease: n = 9), multiple system atrophy (n = 11), progressive supranuclear palsy (n = 16), and of normal controls (n = 13). Brain of a patient with familial Parkinsonism-dementia due to alpha-synuclein locus triplication (as positive control) showed increased membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein levels with abundant high molecular weight immunoreactivity. In multiple system atrophy, a massive increase in 17 kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein was observed in highly pathologically affected regions, including putamen (+1760%, range +625-2900%), substantia nigra [+1000% (+356-1850%)], and white matter of internal capsule [+2210% (+430-6830%)] together with numerous high molecular weight species. Levels of 17 kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein were only modestly increased in less affected areas (cerebellar cortex, +95%; caudate, +30%; with both also showing numerous high molecular weight species) and were generally normal in cerebral cortices. In both Parkinson's disease and progressive supranuclear palsy, membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein levels were normal in putamen and frontal cortex whereas a trend was observed for variably increased 17 kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein concentrations [+184% (-60% to +618%)] with additional high molecular weight species in Parkinson's disease substantia nigra. No obvious correlation was observed between nigral membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein accumulation and Lewy body density in Parkinson's disease. Two progressive supranuclear palsy cases had membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein accumulation in substantia nigra similar to multiple system atrophy. Several Parkinson's disease patients had very modest high molecular weight membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein accumulation in putamen. Levels of 17-kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein were generally positively correlated with those of high molecular weight membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein and there was a trend for a positive correlation between striatal dopamine loss and 17-kDa membrane-associated, sodiu...

Spastic paraplegia is not widely recognized to occur in dopa-responsive dystonia (DRD). The authors found a compound heterozygote for novel mutations of the human tyrosine hydroxylase (TH) gene (TH). The patient was initially diagnosed as having spastic paraplegia, but responded completely to levodopa therapy. Exercise-induced stiffness in the patient's father, who had a TH deletion, also responded to levodopa. The data expand the clinical spectrum of TH deficiency and suggest that TH mutations may account for some patients with DRD simulating spastic paraplegia.

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Because genetic defects relating to the ubiquitin-proteasome system were reported in familial parkinsonism, we evaluated proteasomal function in autopsied brains with sporadic Parkinson's disease. We found that proteasome peptidase activities in a fraction specific to the proteasome were preserved in five brain areas (including the striatum) of Parkinson's disease where neuronal loss is not observed. Striatal protein levels of two proteasome subunits were normal in Parkinson's disease but reduced mildly in disease controls (multiple system atrophy). Our brain data suggest that a systemic, global disturbance in the catalytic activity and degradation ability of the proteasome itself is unlikely to explain the cause of Parkinson's disease.

Human lactoferrin (hLf) has been shown to interact with cells from the Caco-2 human small intestinal cell line. There currently is little information on the molecular details of its interaction. As a first step toward detailed characterization of this interaction, we used a series of Lf chimeras to analyze which part of Lf is responsible for the interaction with Caco-2 cells. Recombinant chimeric proteins consisting of segments of human Lf (hLf) and bovine transferrin (bTf) were produced in a baculovirus-insect cell system, and purified by a combination of cation exchange chromatography and immobilized bTf antibody affinity chromatography. Each chimera was labeled with a green fluorescent dye to monitor its interaction with Caco-2 cells. Similarly the intestinal Lf receptor (LfR), also known as intelectin, was probed with an anti-LfR antibody that was detected with a secondary antibody conjugated with red-color fluorescent dye. The results demonstrated that chimeric proteins containing the N-lobe or the N 1.1 subdomain of Lf bound equally as well as intact Lf to Caco-2 cells. Confocal microscopy analysis revealed that these proteins, along with the LfR, were internalized and targeted to the nucleus. These results indicate that the N1.1 subdomain of human Lf is sufficient for binding, internalization and targeting to the nucleus of Caco-2 cells.Lactoferrin (Lf) is a single-chain, iron-binding glycoprotein that is abundant in milk of some species such as humans, rhesus monkeys, pigs, and mice. It has been suggested to facilitate iron absorption in infants [1]. Lf is also found in high concentration in most exocrine secretions and in the secondary granules of neutrophils, from which it is released following activation of these cells [2,3]. A growing body of evidence supports the role of Lf in a variety of biologically important activities such as defending against a variety of pathogens, modulating the immune system, and stimulating cell proliferation [1]. However, there is little information available on the molecular mechanisms by which Lf mediates these physiological effects. Trans-activation of various genes, such as activation of AP-1 through the JNK and p38 MAPK pathways [4], may be induced by binding of Lf to DNA [5,6], but the signal for nuclear localization of Lf and whether endogenous or external Lf trans-activates gene expression are still not clear. Recently, delta-Lf, a cytoplasmic Lf isoform that is the product of alternative splicing of the Lf gene, was found to provoke anti-proliferative effects and cell cycle arrest in the S phase [7]. Delta-Lf enters the nucleus, and binds to the Skp1 (S-phase kinase-associated protein 1) promoter, suggesting that delta-Lf may regulate cell cycle progression by increasing Skp1 gene expression [8]. The nuclear localization signal sequence of delta-Lf was identified in the Clobe. For exogenous Lf exerting any effects on the cell, interaction with a specific receptor is likely to be involved. This study was initiated to probe the interaction between Lf and th...