Covalent structural changes in unfolded GroES that lead to amyloid fibril formation detected by NMR: insight into intrinsically disordered proteins.

Co-chaperonin GroES from Escherichia coli works with chaperonin GroEL to mediate the folding reactions of various proteins. However, under specific conditions, i.e. the completely disordered state in guanidine hydrochloride, this molecular chaperone forms amyloid fibrils similar to those observed in various neurodegenerative diseases. Thus, this is a good model system to understand the amyloid fibril formation mechanism of intrinsically disordered proteins. Here, we identified a critical intermediate of GroES in the early stages of this fibril formation using NMR and mass spectroscopy measurements. A covalent rearrangement of the polypeptide bond at Asn(45)-Gly(46) and/or Asn(51)-Gly(52) that eventually yield ß-aspartic acids via deamidation of asparagine was observed to precede fibril formation. Mutation of these asparagines to alanines resulted in delayed nucleus formation. Our results indicate that peptide bond rearrangement at Asn-Gly enhances the formation of GroES amyloid fibrils. The finding provides a novel insight into the structural process of amyloid fibril formation from a disordered state, which may be applicable to intrinsically disordered proteins in general.

Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding.

OBJECTIVES: Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. METHODS: Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. RESULTS: Gene expression analysis revealed that Ire1ß was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca(2+)-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. CONCLUSIONS: The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery.

High-resolution nuclear magnetic resonance spectroscopic study of metabolites in the cerebrospinal fluid of patients with cervical myelopathy and lumbar radiculopathy.

There have been few reports describing substances related to oxidative and intermediary metabolism in the cerebrospinal fluid (CSF) in patients with spinal degenerative disorders. This study investigated whether the concentrations of metabolites in the CSF differed between patients with spinal degenerative disorders and controls, and whether the concentrations of these metabolites correlated with the severity of symptoms. CSF samples were obtained from 30 patients with cervical myelopathy (Group M), 30 patients with lumbar radiculopathy (Group R), and 10 volunteers (control). Metabolites in these CSF samples were measured by nuclear magnetic resonance spectroscopy. There were no differences in the concentrations of lactate, alanine, acetate, glutamate, pyruvate, or citrate between Groups M and R, between Group M and the control, or between Group R and the control. In Group M, neither symptom duration nor the Japanese Orthopaedic Association score correlated with the concentration of any metabolite. In Group R, the symptom duration positively correlated with the concentration of lactate, glutamate, and citrate in CSF. The duration of nerve root block showed a negative correlation with the concentrations of acetate in CSF of the patients in Group R. In patients with lumbar radiculopathy, there is a possibility of increased aerobic metabolic activity or decreased gluconeogenic activity in patients with shorter symptom duration, and increased aerobic metabolic activity in patients with severe inflammation around a nerve root.

Up-regulation in the expression of renin gene by the influence of aluminium.

The excretion of aluminium in urine was significantly increased after intake of analgesics containing aluminium, confirming increased absorption and hence exposure to aluminium with such medication. The effect of aluminium on the kidney was further investigated by study of gene expression in mice. After a single dose of aluminium, an up-regulation of renin gene was found by DNA sequencing of the products of differential display analysis. The up-regulation of renin was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments in the dose dependent treatments and the time course observation after aluminium citrate injection. The up-regulation of the renin expression by aluminium is a strong indication of the influence of aluminium on the renin-angiotensin-aldosterone-system, resulting in possible induction of essential hypertension.

(13)C heteronuclear NMR studies of the interaction of cultured neurons and astrocytes and aluminum blockade of the preferential release of citrate from astrocytes.

Citrate has been identified as a major tricarboxylic acid (TCA) cycle constituent preferentially released by astrocytes. We undertook the present study to examine further the nature of metabolic compartmentation in central nervous system tissues using (13)C-labeled glucose and to provide new information on the influence of aluminum on the metabolic interaction between neurons and astrocytes. Metabolites released into the culture medium from astrocytes and neuron-astrocyte coculture, as well as the perchloric acid extracts of the cells were analyzed using 2D (1)H and (13)C NMR spectroscopy. Astrocytes released citrate into the culture medium and the released citrate was consumed by neurons in coculture. Citrate release by astrocytes was blocked in the presence of aluminum, with progressive accumulation of citrate within the cells. We propose citrate supply is a more efficient energy source than lactate for neurons to produce ATP, especially in the hypoglycemic state on account of it being a direct component of the TCA cycle. Astrocytes may be the cellular compartment for aluminum accumulation as a citrate complex in the brain.

Aluminum induces tau aggregation in vitro but not in vivo.

Etiological studies suggest that aluminum (Al) intake might increase an individual's risk of developing Alzheimer's disease (AD). Biochemical analysis data on the effects of Al, however, are inconsistent. Hence, the pathological involvement of Al in AD remains unclear. If Al is involved in AD, then it is reasonable to hypothesize that Al might be involved in the formation of either amyloid plaques or neurofibrillary tangles (NFTs). Here, we investigated whether Al might be involved in NFT formation by using an in vitro tau aggregation paradigm, a tau-overexpressing neuronal cell line (N2a), and a tau-overexpressing mouse model. Although Al induced tau aggregation in a heparin-induced tau assembly assay, these aggregates were neither thioflavin T positive nor did they resemble tau fibrils seen in human AD brains. With cell lysates from stable cell lines overexpressing tau, the accumulation of SDS-insoluble tau increased when the lysates were treated with at least 100 muM Al-maltolate. Yet Al-maltolate caused illness or death in transgenic mice overexpressing human tau and in non-transgenic littermates well before the Al concentration in the brain reached 100 muM. These results indicate that Al has no direct link to AD pathology.

Some aspects of astroglial functions and aluminum implications for neurodegeneration.

The present decade had witnessed an unprecedented attention focused on glial cells as a result of their unusual physiological roles that are being unraveled. It is now known that, rather than being a mere supporter of neurons, astroglia are actively involved in their modulation. The aluminum hypothesis seems to have been laid to rest, probably due to contradictory epidemiological reports on it as a causative factor of neurodegenerative diseases. Surprisingly, newer scientific evidences continue to appear and recent findings have implicated astrocytes as the principal target of its toxic action. In view of the likely detrimental effects of the interaction between these two infamous partners in neuroscience on neurons and nervous system, we have reviewed some aspects of glia-neuron interaction and discussed the implications of aluminum-impaired astrocytic functions on neurodegeneration. Because sporadic causes still account for the majority of the neurodegenerative diseases of which Alzheimer's disease is the most prominent, it has been suggested that neurotoxicologists should not relent in screening for the environmental agents, such as aluminum, and that considerable attention should be given to glial cells in view of the likely implications of environmental toxicants on their never-imagined newly reported roles in the central nervous system (CNS).

Accumulation of aluminum by primary cultured astrocytes from aluminum amino acid complex and its apoptotic effect.

Aluminum salts or doses that are unlikely in the human system have been employed in toxicity studies and much attention had been focused on the secondary target (neurons) of its toxicity rather than the primary target (astroglia). In order to address these issues, we have investigated the uptake and apoptotic effects of aluminum amino acid complex on primary cultured astrocytes because these are fundamental in understanding the mechanism of aluminum neurotoxicity. Aluminum solubilized by various amino acids was differentially internalized by astrocytes (glycine>serine>>glutamine>>glutamate), but aluminum was not internalized from citrate complex following 24 h of exposure. Inhibition of glutamine synthetase, by methionine sulfoximine (MSO), enhanced the uptake of aluminum from various amino acid complexes within 8 h except from glutamine complex. Blockade of selective GLT-1 (EAAT2) and GlyT1, as well as nonspecific transporters, did not inhibit or had no effect on uptake of aluminum in complex with the corresponding amino acids. Ouabain also failed to inhibit uptake of aluminum complexed with glycine. Pulse exposure to aluminum glycinate in the absence or presence of MSO caused apoptosis in over 25% of primary cultured astrocytes, and apoptotic features such as chromatin condensation and fragmentation became evident as early as 3 days of culture in normal medium. Lower doses (as low as 0.0125 mM) also caused apoptosis. The present findings demonstrate that aluminum solubilized by amino acids, particularly glycine, could serve as better candidate for neurotoxicity studies. Citrate may be a chelator of aluminum rather than a candidate for its cellular uptake. Amino acid transporters may not participate in the uptake of aluminum solubilized by their substrates. Another pathway of aluminum internalization may be implicated in addition to passive diffusion but may not require energy in form of Na+/K+-ATPase. Impaired astrocyes' metabolism can aggravate their accumulation of aluminum and aluminum can compromise astrocytes via apoptosis. Thus, loss of astrocytic regulatory and supportive roles in the central nervous system (CNS) may be responsible for neurodegeneration observed in Alzheimer's disease.

Metals accelerate production of the aberrant splicing isoform of the presenilin-2.

Oxidative stress is a major risk factor for Alzheimer's disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin-2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal-induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre-mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.

Automated graphic image generation system for effective representation of infectious disease surveillance data.

Infectious disease surveillance schemes have been established to detect infectious disease outbreak in the early stages, to identify the causative viral strains, and to rapidly assess related morbidity and mortality. To make a scheme function well, two things are required. Firstly, it must have sufficient sensitivity and be timely to guarantee as short a delay as possible from collection to redistribution of information. Secondly, it must provide a good representation of the results of the surveillance. To do this, we have developed a database system that can redistribute the information via the Internet. The feature of this system is to automatically generate the graphic images based on the numerical data stored in the database by using Hypertext Preprocessor (PHP) script and Graphics Drawing (GD) library. It dynamically displays the information as a map or bar chart as well as a numerical impression according to the real time demand of the users. This system will be a useful tool for medical personnel and researchers working on infectious disease problems and will save significant time in the redistribution of information.

Direct observation of 3-keto-valproate in urine by 2D-NMR spectroscopy.

BACKGROUND: It is known that valproate and its metabolites cause hepatotoxicity. The drug monitoring of valproate is important to determine an effective dose to keep an appropriate concentration in blood. METHODS: In a 2-dimensional (2D)-NMR spectrum of double quantum filtered correlation spectroscopy (DQF-COSY), clear correlation peaks were ascertained to be due to 3-keto-valproate, which was a beta-oxidation product of valproate. RESULTS: A predominant metabolite of valproate was observed by proton NMR spectroscopy in the crude urine of a particular patient with metabolic disorder. The assignment of the signals was determined by synthesized 3-keto-valproic acid ethyl ester. The concentration of 3-keto-valproate in the urine was calculated to be 631 microg/mg creatinine by the integration of the peak of the isolated triplet methyl protons of C(5) at 1.016 ppm. CONCLUSION: Although the NMR spectra of crude urine of the patients who took valproate were usually complicated with many metabolites, the signals of 3-keto-valproate in a DQF-COSY spectrum of the urine of patients were easily connected according to the present assignment. The NMR analysis of the urine of patients who are prescribed valproate is useful for therapeutic drug monitoring and for checking the compliance of the patients.

Development of computerized Kana Pick-out Test for the neuropsychological examination.

The Kana Pick-out Test, which was developed in Japan and done with paper and pencil, is said to be suitable for inspecting higher-order brain function and to be a good method for screening persons with mild or slight dementia. We have developed a computerized version of the Kana Pick-out Test, which runs on a stand-alone computer, intended to be utilized for mass screening and self-administration. The program was developed with Microsoft Visual Basic 6.0 and runs under the Windows operating system on any IBM PC compatible computer. In this study, all subjects could use the system by interacting with the computer and it was found that the system seemed to have the capability of detecting cognitive status equal to the paper-based Kana Pick-out Test. Besides this, we developed a network-based Kana Pick-out game software which was intended to attract user's notice. The game program was written in JAVA language and runs on a web-browser supporting JAVA on any operating system. The program, a so called applet, is located on our web site (http://environ.med.tottori-u.ac.jp) and anyone can use the applet by accessing our homepage.

Heavy metal analysis of groundwater from Warri, Nigeria.

The levels of some ions of heavy metals known to be associated with petroleum industry operations, including Pb, Ni, V, Cr, Cd, Zn and Fe, were studied in untreated groundwater from Warri area, Nigeria by atomic absorption spectroscopy. Warri area is characterized by petroleum industry activities including a Refinery. With this in mind, the residential area was divided into Effurun junction, Waterside Ekpan village and the Refinery's vicinity. The concentrations of Pb, Ni and Fe measured (in mg x l(-1)) in the groundwater samples of all areas studied ranged from 0.06 to 0.44, 0.008 to 0.19 and 0.315 to 2.753 respectively, while V, Cr, Zn and Cd were present in very low concentrations, 0-0.85 x 10 (-3). The levels of Pb, Ni and Fe exceeded the threshold limits (0.01, 0.02 and 0.3 mg x l(-1), respectively) set by the WHO health-based guideline for drinking water and this could portend environmental hazards.