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Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of α-synuclein filaments. αB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of αB-crystallin in GCIs in MSA brains. We also examined the influence of αB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed αB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that αB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for αB-crystallin. Proteasomeinhibited cells transfected with GFP-tagged α-synuclein resulted in ubiquitin- and αB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that αB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.

Streptozotocin (STZ) and alloxan (ALX) are potent diabetogens in different species of laboratory animals. Here, we describe differential in vitro effects of STZ and ALX on β-cell molecules that are essential for glucose transport and metabolism, the glucose transporter 2 (GLUT2) and glucokinase (GK), respectively. Incubation of isolated pancreatic islets of C57 BL/6 mice with STZ or ALX for 30 min resulted in a concentration-dependent gradual loss of β-cell function as determined by basal and D-glucose (D-G)-stimulated insulin release. ALX concentration-dependently reduced the mRNA expression of GLUT2 and GK and the effect on GLUT2 was more marked. STZ, in contrast, did not affect the mRNA expression of GLUT2 and GK, but concentration-dependently reduced the GLUT2 protein expression. Both STZ and ALX failed to affect the mRNA expression of proinsulin and of β-actin. The deleterious effects of STZ and ALX were not due to β-cell loss, because the total RNA yields and protein contents as well as the proinsulin mRNA expression in isolated islets of the differentially treated islets did not differ significantly from controls. Furthermore, islets that had been exposed to STZ or ALX responded to the non-glucose secretagogue arginine in a pattern comparable to that of solvent-treated cultures. When preincubating islet cultures with either D-G or its chemically closely related analogue 5-thio-D-glucose (5-T-G), different effects were obtained after treatment with either ALX or STZ. Thus, preincubation with 5-T-G protected the cultures from STZ-induced GLUT2 protein reduction, whereas D-G failed to do so. Preincubation with D-G, however, protected the cultures from ALX-induced reduction of GLUT2 and GK mRNA expression, whereas 5-T-G, at best, exerted a modest protection against ALX at a concentration of 1 mmol/l. Apparently, in vitro, GLUT2 protein is a key target molecule for STZ, while GLUT2 mRNA and GK mRNA are target molecules for ALX.

Older patients who die with Parkinson's disease (PD) have fewer pigmented neurons in the locus coeruleus and fewer substance P-containing neurons in mesopontine tegmental nuclei. We analyzed two other medullary nuclei, the dorsal vagal nucleus and the hypoglossal nucleus, in eight PD patients and six normal controls by counting neurons in serial Nissl stained sections to determine the relationship between age at death and cell loss in these nuclei. PD-related neurodegenerative changes (Lewy bodies and neuronal loss) were present only in the dorsal vagal nucleus (13,637 ± 1,323 neurons in PD, 24,885 ± 1,157 in normal controls). Cells in the intermediate rostrocaudal part of the nucleus were most severely affected. There was a significant correlation between loss of vagal neurons and age at death in PD patients. No age-related cell loss was present in the dorsal vagal nucleus of normal brains, or in the hypoglossal nucleus in either PD or normal brains. These results confirm that age-related cell death depends on whether or not there is coexistent PD.

Ligninolytic fungi are unique among eukaryotes in their ability to degrade polycyclic aromatic hydrocarbons (PAHs), but the mechanism for this process is unknown. Although certain PAHs are oxidized in vitro by the fungal lignin peroxidases (LiPs) that catalyze ligninolysis, it has never been shown that LiPs initiate PAH degradation in vivo. To address these problems, the metabolism of anthracene (AC) and its in vitro oxidation product, 9,10-anthraquinone (AQ), was examined by chromatographic and isotope dilution techniques in Phanerochaete chrysosporium. The fungal oxidation of AC to AQ was rapid, and both AC and AQ were significantly mineralized. Both compounds were cleaved by the fungus to give the same ring-fission metabolite, phthalic acid, and phthalate production from AQ was shown to occur only under ligninolytic culture conditions. These results show that the major pathway for AC degradation in Phanerochaete proceeds AC----AQ----phthalate + CO2 and that it is probably mediated by LiPs and other enzymes of ligninolytic metabolism.

Abstract: Ubiquitin-positive Lewy neurites and Lewy bodies are found in idiopathic Parkinson's disease (PD) and diffuse Lewy body disease (DLBD). We found that, in three patients with PD and one with DLBD, microtubule-associated protein 5 (MAP5) immunostaining was consistently present in both Lewy neurites and Lewy bodies throughout the brainstem and forebrain regions affected in the disease. In contrast, other cytoskeletal markers (neurofilaments and MAP2) could be demonstrated in only a small fraction of Lewy bodies and neurites. Confocal microscopy demonstrated that MAP5 immunolabeling was located around the perimeter of the ubiquitin-positive labeling which occupied the central region of the neurite and Lewy body, with some overlap between MAP5 and ubiquitin staining. In contrast, in those Lewy bodies and neurites immunopositive for phosphorylated and non-phosphorylated neurofilament proteins, the neurofilament labeling was quite peripheral to the ubiquitin staining, with little or no overlap. Our results suggest MAP5 is more closely associated with the ubiquitinated proteins of Lewy bodies and neurites than other cytoskeletal proteins.

SUMMARY Substance P immunoreactive (SP+) neurons were analysed quantitatively in serial sections of the mesopontine tegmentum in 6 patients with idiopathic Parkinsons disease and 5 age-matched normal controls. In the tegmentum of the Parkinsons disease brains many SP+ neurons contained swollen, twisted neuronal processes as well as Lewy bodies. There were significant reductions in the total number of SP+neurons in the pedunculopontine tegmental nucleus (loss 43%), in the laterodorsal tegmental nucleus (loss 28%), in the oral pontine reticular nucleus(loss 41%) and in the median raphe nucleus (loss 76%). It was the large SP+ (>20μm) neurons that were particularly affected. In our control group we did not document a significant relationship between age at death and number of SP+neurons in these tegmental nuclei or between age at death and number of pigmented neurons in the locus coeruleus. In contrast, in patients with Parkinsons disease, there was a strong inverse relationship between age at death and numbers of SP+ and pigmented neurons. Our findings suggest an interaction between the pathophysiological mechanisms initiated by Parkinsons disease and other processes related to ageing. Since tegmental SP+neurons are affected by the primary pathological processes underlying Parkinsons disease as severely as catecholamine-synthesizing neurons are affected, theories of pathogenesis and therapeutic strategies in Parkinsons disease will need to take into account the involvement of these SP+neurons.

Characteristic ubiquitin-positive, tau-negative, degenerating neurites were present in brainstem regions known to be involved in idiopathic Parkinsons disease. Corresponding changes were entirely absent from controls and from the brainstems obtained from patients who had died with Alzheimers disease, motor neuron disease and multiple system atrophy. In Parkinsons disease cases degenerating neurites were particularly striking in the dorsal motor nucleus of the vagus. In this nucleus the density of degenerating neurites was inversely related to the duration of Parkinsons disease symptoms. Some ubiquitinpositive degenerating neurites also contained neurofilament immunoreactivity. However, confocal microscopy revealed that ubiquitin and neurofilament reactivities were located in separate regions of the degenerating neurite, suggesting that proteins other than neurofilaments may be important in the process of ubiquitination. The demonstration of ubiquitinpositive degenerating neurites in routinely prepared paraffinembedded material, particularly in the dorsal motor nucleus of the vagus, could become diagnostically useful in those Parkinsons disease cases in which Lewy bodies are difficult to find. Demonstration of extensive ubiquitin-positive degenerating neurites might provide a clue to disease activity at the time of death.

Identification of human parasympathetic preganglionic neurons in pontomedullary regions has been largely based on studies using cholinesterase histochemical procedures, and so far there is no adequate account of the location of these cells. Nitric oxide synthase (NOS) is present in brainstem parasympathetic preganglionic salivatory neurons in the rabbit (Zhu et al., 1996). In the present study we have used histochemical and immunohistochemical staining for NOS to examine possible preganglionic parasympathetic neurons in the human brainstem. We examined, in five human brains, the distribution, through the caudal pons and rostral medulla, of NOS-positive neurons in serial sections stained with NADPH diaphorase for histochemistry, and with antibodies against neuronal NOS peptide for immunohistochemistry. In scattered pontomedullary regions (rostral to the dorsal motor nucleus and the nucleus ambiguus) known to contain parasympathetic preganglionic neurons in animals, we observed groups of NOS-positive neurons which correspond in morphology and distribution with NOS-positive parasympathetic preganglionic neurons in rabbits. These neurons are probably parasympathetic preganglionic neurons in the human brainstem, involved in the control of lacrimation, salivation, oral and nasopharyngeal secretion, as well as the control of the dilation of extra-and intracranial blood vessels.