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Biomedical subjects

F Watt

Publications and source records attributed to F Watt.

At least 37 records · Page 2Linked to original sources
PubMed1996

Nuclear microscope analysis in Alzheimer's and Parkinson's disease: A review.

The scanning nuclear microprobe (nuclear microscope) is becoming a powerful instrument for the accurate measurement of minor and trace elements in biological tissue. Using the simultaneously applied techniques of Scanning Transmission Ion Microscopy (STIM) to image features in the tissue, Particle induced X-ray emission (PIXE) to measure trace element concentrations, and Rutherford Backscattering Spectrometry (RBS) to characterize the tissue matrix, accurate elemental analysis at the parts per million level can be obtained for most elements. This review describes briefly the results obtained using the nuclear microscope for the elemental analysis of Alzheimer's and Parkinson's tissue. In Alzheimer's disease (AD) the identification and subsequent analysis of neuritic plaque cores in unstained tissue, yielded an absence of aluminium at the limit of 15 parts per million. Previous analyses involving stained sections were prone to misinterpretation due to aluminium contamination from the staining procedures. Elemental iron, calcium, phosphorus and sulphur were elevated both in the plaques and the AD background tissue compared to age matched controls. Preliminary analyses of neurofibrillary tangles stained with toluidine blue showed increased levels of calcium, although the staining procedure may have distorted the results due to element redistribution. In Parkinson's disease (PD) nuclear microscope studies have concentrated on measurements of iron in the substantia nigra (SN) region of the brain; iron was observed to be elevated by a factor 2 in MPTP induced Parkinsonism in African Green monkeys, and by a factor of 1.25 in 6-OHDA induced Parkinsonism in Sprague Dawley rats. These studies are consistent with other studies showing a general increase in the concentrations of iron associated with PD, and support the theory that iron mediated free radical production may enhance or accelerate the degeneration of dopaminergic cells through oxidative stress.

Alzheimer Disease↗
PubMed1996

Elemental changes in atherosclerotic lesions using nuclear microscopy.

The roles of iron and other trace metals in the aetiology of heart and brain diseases has been a subject of keen research because of the ability of metal ions to participate in reactions involving free radicals, which have been implicated in many of these diseases. Unstained freeze dried tissue sections from the aorta of New Zealand white rabbits fed with a 1% cholesterol diet for 12 weeks were scanned with a 2 MeV proton beam using the National University of Singapore nuclear microscope facility. Results from 6 test and 4 control rabbits show that there is an average of seven-fold increase in iron and an average of nearly two-fold increase in phosphorus in the atherosclerotic lesion compared with healthy tissue. The increase in iron adds weight to the hypothesis that iron-catalyzed free radical reactions may be associated with the development of atherosclerosis. A depletion of other elements analyzed was seen in the lesion. Elemental mapping also showed the occurrence of granules rich in sodium, chlorine and potassium at the interface between lesioned and non-lesioned tissue.

Animals↗
PubMed1996

A review of nuclear microscopy and applications in medicine.

Nuclear Microscopy, the extraction of analytical information from microscopic regions of a sample using a scanning focused high energy ion beam, has been increasing in popularity recently, despite its technical complexity. The three ion beam related techniques Particle Induced X-ray Emission (PIXE), Rutherford Backscattering Spectrometry (RBS) and Scanning Transmission Ion Microscopy (STIM) can be carried out simultaneously at sub-micron spatial resolutions, and provide structural and quantitative elemental analysis down to the parts per million levels of analytical sensitivity. These techniques are extremely useful for measuring any imbalances in trace elements, including metal ions, in localised regions of biological tissue, and as such can provide unique information on many diseases. In this paper we briefly describe the nuclear microscope and its related ion beam techniques, and briefly review recent work carried out using the nuclear microscope into the degenerative diseases Alzheimer's disease, Parkinson's disease and atherosclerosis.

Alzheimer Disease↗
PubMed1995

Analysis of copper and lead in hair using the nuclear microscope; results from normal subjects, and patients with Wilson's disease and lead poisoning.

We have recently developed a technique utilizing the nuclear microscope to analyse accurately the trace element content of hair at concentrations of ppm and to differentiate between the levels within hair and those due to surface contamination. Hair was analysed from four patients with Wilson's disease (systemic copper overload), one patient with sub-acute lead poisoning, and three control subjects, and the concentrations and distributions of lead and copper across the hair shafts were shown. However, there was no increase in the copper content of hair from patients with Wilson's disease compared with those of normal subjects, indicating that the high copper levels of many tissues in this disease are not reflected in hair. In contrast, hair samples from the patient with lead poisoning showed maximum concentrations of 14 (+/-2) micrograms g-1, compared with normal levels of less than 1 micrograms g-1. Further, the composition of the hairs along the shaft reflected the temporal changes of lead levels in the patient during lead elimination.

Adolescent↗
PubMed1995

Assessment of toxic metal exposure following the Camelford water pollution incident: evidence of acute mobilization of lead into drinking water.

Following the incident of acidic pollution of water by aluminium sulfate centred around Camelford in July 1988, we have carried out a retrospective analysis of the mobilization of toxic metals to residents of the area. An advanced nuclear technique was used to measure trace levels of elements within hair, thus, avoiding surface contamination. In contrast to controls, lead, but no other toxic metals, was consistently found within sections of hair that dated to mid-1988 from four residents; they must, therefore, have consumed this metal around the time of the incident. The source of this lead was probably local water pipe residue, and this was found on analysis to have a matrix specific to such soft-water areas that, prior to the incident, had slowly accumulated certain toxic metals such as cadmium and uranium and particularly lead. Lead is mobilized from such residues by acidic water and could, therefore, have heavily contaminated mains water after the incident. However, analyses of residents' plasma and whole blood, and of urine following a lead-chelation test, showed no evidence of either long-term increased body burdens of toxic metals or depletion of essential elements. In addition, we found no evidence of continued poor water quality in the area. In conclusion, during a short period following the pollution, some residents who consumed mains water would have been acutely exposed to lead and other toxic metals. Prediction of the scale of metal exposure to individuals was not possible owing to heterogeneity of the water distribution network, but long-term effects to residents from lead are not anticipated.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1994

Increased iron in the substantia nigra compacta of the MPTP-lesioned hemiparkinsonian African green monkey: evidence from proton microprobe elemental microanalysis.

The association of free radicals and particularly free iron in the pathogenesis of idiopathic Parkinson's disease and MPTP-induced parkinsonism remains controversial. Whereas the actual cause of dopamine cell death in the substantia nigra compacta (SNc) remains unknown, disturbances in lipid peroxidation and subsequent mitochondrial and cell membrane disruption has been demonstrated. In a genetically susceptible host, abnormal elimination of oxygen and trace metal free radicals may further damage dopamine cells. Using a unilaterally MPTP-treated African Green monkey, which showed obvious contralateral hemiparkinsonism, the total free iron concentration was measured. Iron, Fe2+ and Fe3+, but not other trace elements, was significantly elevated in the SNc compared with the opposite unlesioned side, which was similar to separate control animals. Iron content in the SNc, periaqueductal gray area, and crus cerebri was 228-270 ppm. Normal control SNc was 285 (+/- 59) ppm, whereas iron levels of 532 (+/- 151) ppm were found in the MPTP-lesioned SNc. These animals were drug naive and not on long-term levodopa maintenance. Proton microprobe elemental analysis was matched against adjacent immunocytochemically stained tissue slices to ensure the cells studied were in the SNc. Iron was found not only in the degenerating dopamine cells themselves but also in the surrounding matrix and glial cells. Whether free iron that is not bound to neuromelanin is responsible for dopamine cell death as suggested by these experiments remains to be proved.

Animals↗
PubMed1993

Specific cleavage of transcription factors by the thiol protease, m-calpain.

The intracellular nonlysosomal calcium-dependent cysteine protease, m-calpain, is shown to specifically cleave the bHLHzip transcription factor USF leaving the binding and dimerisation domains intact. The resultant protein is capable of efficient DNA binding but is no longer able to activate transcription. A surprisingly high proportion of other transcription factors tested, AP1 (c-Fos/c-Jun), Pit-1, Oct-1, CP1a and b, c-Myc, ATF/CREB, AP2 and AP3 but not Sp1, were similarly cleaved by m-calpain to produce specific partial digestion products. These properties make m-calpain a particularly useful protease for proteolytic studies of transcription factors and also raise the possibility that m-calpain may be involved in vivo in regulation of turnover or transcriptional activity of a number of transcription factors.

Base Sequence↗
PubMed1992

Absence of aluminium in neuritic plaque cores in Alzheimer's disease.

Controversy exists over whether aluminium has a role in the aetiology of Alzheimer's disease. Alzheimer's disease is neuropathologically characterized by the occurrence of a minimum density of neurofibrillary tangles and neuritic plaques in the hippocampus and the association cortex of the brain. The purported association of aluminium with Alzheimer's disease is based on: (1) the experimental induction of fibrillary changes in the neurons of animals by the injection of aluminium salts into brain tissue; (2) reported detection of aluminium in neuritic plaques and tangle-bearing neurons; (3) epidemiological studies linking aluminium levels in the environment, notably water supplies, with an increased prevalence of dementia; and (4) a reported decrease in the rate of disease progression following the administration of desferroxamine, an aluminium chelator, to clinically diagnosed sufferers of Alzheimer's disease. Here we use nuclear microscopy, a new analytical technique involving million-volt nuclear particles, to identify and analyse plaques in postmortem tissue from patients with Alzheimer's disease without using chemical staining techniques and fail to demonstrate the presence of aluminium in plaque cores in untreated tissue.

Aluminum↗
PubMed1992

Binding of proteins from embryonic and differentiated cells to a bidirectional promoter contained within a CpG island.

We have analysed binding sites of nuclear protein factors to a CpG island (HTF9), which contains the promoter for a pair of overlapping, divergently-transcribed "housekeeping" genes. Using DNaseI protection assays with extracts from a range of differentiated and undifferentiated cell lines, including mouse embryonic stem (ES) and embryonal carcinoma (EC) cells, we located multiple protein binding sites on HTF9. Most of the sites were outside the defined core promoter and could bind to previously identified transcription factors. These included constitutive, inducible and apparently tissue-specific factors in an extremely asymmetric array relative to the transcription start sites of the two genes. A number of sites showed different binding specificities or affinities in different cell types, including ES cells. However, we found no factors that were specific for both ES and EC cells, and no protein-binding site protected exclusively in undifferentiated embryonic cells.

3T3 Cells↗
PubMed1992

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

5-Methylcytosine↗
PubMed1992

Elemental analysis of neurofibrillary tangles in Alzheimer's disease using proton-induced X-ray analysis.

We have investigated the elemental content of hippocampal slices from normal human brain and from brains of Alzheimer's disease patients by X-ray fluorescence using both electron and proton beam microprobes. The sections have been stained with a dye--toluidine blue--which contains sulphur so that the X-ray fluorescence map can be correlated with known intracellular sites as seen under the light microscope. The results show that associated with neurofibrillary tangles and Hirano bodies (the distinctive internal visual features of cells from Alzheimer's disease patients) there is increased calcium. We cannot confirm that there are peculiarities in the distribution of aluminium in cells.

Alzheimer Disease↗
PubMed1991

Characterization of the human N-ras promoter region.

Overexpression of ras proto-oncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer in order to determine functional regions within the human N-ras promoter. A significant proportion of promoter activity was found to lie within a 439 bp fragment comprising an untranslated exon (exon 1) with the adjacent 5' sequence and a small CpG island. A 109 bp [corrected] fragment at the 5' end of exon 1 was essential for promoter activity, while a 45 bp [corrected] deletion from within this region decreased promoter activity by two-thirds. Unlike the human H-ras and mouse K-ras promoters, the N-ras promoter did not exhibit bidirectional activity. DNAse footprinting of the 439 bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA-binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb and E4TF1). In contrast, four putative Sp1 sites did not footprint. Using purified MLTF and appropriate competitors in gel shift and DNAase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon 1.

Amino Acid Sequence↗
PubMed1990

DNA methylation and specific protein-DNA interactions.

The effect of site-specific CpG methylation on the binding of a HeLa cell transcription factor (MLTF) has been studied. Methylation at a central site within the binding sequence for the factor is found to strongly inhibit binding of MLTF and to inhibit MLTF-dependent transcription in vitro. Methylation of a CpG site only six bases away has no demonstrable effect on binding. When the central CpG is methylated on one strand only, binding of MLTF is partially inhibited. The effects of methylation on the binding of MLTF and on the binding of a limited number of other proteins to DNA demonstrate that methylation in some cases exerts highly specific effects on gene expression. Site-specific demethylation has previously been seen to be associated with the promoter and upstream regions of genes, which suggests that sequence-specific DNA binding proteins that interact with promoters may be involved in the demethylation process. Specific demethylation of a human metallothionein 2A promoter-chloramphenicol acetyl transferase gene construct in mouse L-cells demonstrates that the promoter region sequences of this gene are sufficient to programme their own demethylation.

Animals↗
PubMed1989

Bands, interbands and puffs in native Drosophila polytene chromosomes are recognized by a monoclonal antibody to an epitope in the carboxy-terminal tail of histone H1.

A monoclonal antibody was raised against Drosophila melanogaster histone H1. Immunoscreening of proteolytic cleavage fragments of H1 and of a set of all possible overlapping synthetic octapeptides corresponding to the amino acid sequence of H1, revealed that the antibody recognizes an epitope within the sequence 207VTAAKPKA214 near the centre of the carboxy-terminal tail. This antibody gives positive immunofluorescence over the entire length of native D. melanogaster polytene chromosomes isolated from salivary glands by microdissection at physiological pH and ionic strength. Bands, interbands and puffs are all seen to contain H1. The immunofluorescence over puffs, albeit lower than that over bands and interbands, indicates that chromatin decondensation can occur without complete loss of H1 in these structures. The reaction of the antibody with bands suggests that the segment of the C-terminal tail containing the epitope may be exposed in the condensed 30 nm chromatin filament.

Amino Acid Sequence↗