Identification of the novel HLA-A*02:829 allele by sequencing-based typing.

The novel HLA-A*02:829 allele is likely generated from the recombination of both HLA-A*02:05:01:01 and HLA-A*32:01:01:01 alleles.

Moesin: A novel receptor on NK lymphocytes binds to TOMM40 on K562 leukemia cells initiating cytolysis.

Natural killer lymphocytes (NK cells) are the first line of defense (innate immunity) against viral infections and leukemia since they do not require activation to deliver a lethal hit to infected/aberrant cells. In contrast, T lymphocytes require stimulation by a foreign/neo - antigen, which may take days before they are active against the pathogen (adaptive immunity). A number of receptors on activated NK cells that kill the prototypical leukemia target cell line, K562, have been identified. To date, the receptor(s) by which freshly isolated unstimulated NK cells (naïve, nNK) kill K562 has not been fully elucidated. We provide peptide sequence and immune-blot data from ligand pull down experiments that moesin, a protein that typically links the inner leaf of the plasma membrane to the cytoskeleton, additionally, in NK cells, localizes to the cell surface where it may bind to its ligand, TOMM40 (aka Haymaker, HYMKR), on leukemia cells thereby initiating their destruction. Flow cytometry experiments with a mouse monoclonal antibody (Mab) to a moesin peptide (554 to 565) were performed. Moesin was detected on the surface of CD3-, CD16+nNK cells but was not detected on the surface of freshly isolated unstimulated CD3+, CD16- T cells or CD19+, CD16- B cells from healthy subjects. Moesin, is therefore another marker that distinguishes unstimulated CD3-, CD16+ NK cells from other non-activated lymphocytes. The anti -moesin peptide Mab was highly effective (>95% inhibition) in blocking target cell cytolysis by CD16+ lymphocytes demonstrating that moesin-HYMKR interaction appears to be necessary for most of the observed cell death of K562 caused by unstimulated NK cells.

Camptothecin sensitivity in Werner syndrome fibroblasts as assessed by the COMET technique.

Werner syndrome (WS) is an inherited genetic disease in which individuals display the premature aging of a selected subset of tissues. The disorder results from the loss of function mutations in the wrn gene. Wrn codes for a member of the RecQ helicase family with a unique nuclease domain. There is significant evidence that the role of wrn is to assist in the repair and reinitiation of DNA replication forks that have stalled. Loss of the wrn helicase imposes a distinct set of phenotypes at the cellular level. These include premature replicative senescence (in a subset of cell types), chromosomal instability, a distinct mutator phenotype, and hypersensitivity to a limited number of DNA damaging agents. Unfortunately, most of these phenotypes are not suitable for the rapid assessment of loss of function of the wrn gene product. However, WS cells have been reported to show abnormal sensitivity to the drug camptothecin (an inhibitor of topoisomerase type I). A rapid assay for this sensitivity would be a useful marker of loss of wrn function. The COMET (single-cell gel electrophoresis) assay is a rapid, sensitive, versatile, and robust technique for the quantitative assessment of DNA damage in eukaryotic cells. Using this assay, we have found that a significantly increased level of strand breaks can be demonstrated in WS cells treated with camptothecin compared with normal controls.

Further evidence for a role of the anterior claustrum in epileptogenesis.

The anatomy of the claustrum (CLA) has been well characterized, but its functional role remains uncertain. The results of recent research suggest that the CLA may be part of a network of structures involved in seizure generalization, and we set out to test this idea. To test persistence, seizures were kindled in the anterior CLA. Following a 14-day suspension of kindling, all rats required only one stimulation to evoke a stage 5 seizure. In another experiment, groups of rats received bilateral lesions of the anterior CLA before and after amygdaloid kindling. We found that small lesions of the anterior CLA retard amygdaloid kindling, but do not block the expression of generalized seizures. Lesions produced after amygdaloid kindling resulted in a shorter seizure duration, but had no marked effect on seizure expression. Another group of rats was tested for transfer of kindling between the anterior CLA and contralateral amygdala. We found an asymmetrical transfer of kindling to the CLA from the amygdala wherein amygdaloid kindling facilitated subsequent kindling of the CLA but kindling of the anterior CLA failed to facilitate kindling of the amygdala. The results add support to the notion that the CLA contributes to the development of generalized limbic seizures.

A model for the phenotypic presentation of Werner's syndrome.

Werner's syndrome (WS) is a valuable model of accelerated ageing and results from mutations in a recQ helicase (wrn). WS fibroblasts show a mutator phenotype, replication fork stalling, increased rates of mean telomeric loss and accelerated cellular senescence. Senescence has been proposed as a candidate mechanism for the ageing of mitotic tissue. However, some mitotic tissues (such as the immune system) seem unaffected in WS. Is this evidence against a role for cell senescence in ageing? Two experiments resolve this paradox (i) the demonstration that the abbreviated replicative lifespan of WS fibroblasts can be corrected by the ectopic expression of telomerase and (ii) the demonstration that T cells derived from WS patients have the mutator phenotype characteristic of the disease but show no reduction in replicative potential. Since T cells can upregulate telomerase naturally these findings are consistent with a model in which the only wrn-mediated deletions that have a significant effect on replicative lifespan are those at or near the telomere. These data are thus supportive of a role for senescence in the ageing of the immune system. Emerging data on divisional counting mechanisms have the potential to produce many other apparent WS "paradoxes". Accordingly, we propose a general model for the phenotypic presentation of WS, which includes a modification of the Olovnikov model of telomere erosion. Somewhat unexpectedly, this predicts that accelerated senescence should not be observed in all telomerase-negative WS cell types.

Altered composition of the AP-1 transcription factor in immortalized compared to normal proliferating cells.

We have investigated the expression of the AP-1 transcription factor proteins, fos, fosB, fra1, fra2, jun, junB, junD, using Western blot analysis, in several types of asynchronously proliferating cells. The latter included normal fibroblasts, immortalized but not tumourigenic fibroblasts, and two immortalized tumour cell lines. All cells expressed fos, fra1 and jun proteins and none expressed fosB. There were, however, interesting qualitative differences between the normal fibroblasts and the immortalized cells. Expression of fra2 was difficult to detect in normal cells, but was very evident in all of the immortalized cells. The normal cells only expressed a 44 kDa junB species, whereas the immortalized cells expressed both this and another 34 kDa species. All of the cells expressed the two junD proteins but the smaller 39 kDa species was more prominent in the normal cells, whereas the larger 44 kDa protein was more prominent in the immortalized cells. These data indicate that immortalized cells are not simply cells in which the ageing process has been prevented or reversed, but instead exhibit additional characteristics to those associated with young normal cells.

Altered composition and DNA binding activity of the AP-1 transcription factor during the ageing of human fibroblasts.

We have investigated the expression of AP-1 transcription factor proteins during the in-vitro ageing of human fibroblasts. The numbers of these cells that are in the cell cycle gradually decreases up to 45 cumulative population doublings (cPD), thereafter the decline is steeper, until almost all cells enter a post-mitotic state by 60 cPD. We observed that a 34 kd junB species began to replace the 44 kd junB species after 41 cPD. This was followed, after 44 cPD, by a loss of fra1 and both junD species. After 49 cPD there was a gradual decline in the levels of fos and jun proteins, but disproportionately, so that the fos/jun protein ratio also declined. Although fos and jun proteins were still clearly present at 60 cPD, utilisation of the AP-1 DNA consensus sequence could not be demonstrated after 54 cPD. These data indicate that significant changes occur in the composition of the AP-1 transcription factor during ageing, but also that alterations in its DNA binding activity may involve other factors.

1H-NMR analysis of microbial-derived organic acids in primary root carious lesions and saliva.

In addition to lowered pH values, the molecular profile and concentrations of microbial-derived organic acids in carious dentin are important demineralization parameters involved in the induction, development and progression of dental caries. High-resolution proton ((1)H) NMR spectroscopy was employed to examine the organic acid status of primary root carious lesions. (1)H-NMR analysis of post-neutralized perchloric acid extracts of active carious lesions revealed that at an operating frequency of 600 MHz, the (1)H-NMR-detectable organic acid composition of carious dentin samples (mean molecular percentage content +/- standard error; the mean molecular percentage content is defined here as the mean of the concentration of each (1)H-NMR-visible organic acid/anion expressed as a percentage of total (1)H-NMR-detectable organic acid/anion level in each sample) was acetate 51 +/- 2%, formate 37 +/- 2%, lactate 5 +/- 1%, propionate 3 +/- 0.8%, pyruvate 2.4 +/- 0.3%, n-butyrate 1.2 +/- 0.2%; succinate 0.1 +/- 0.1%; iso-butyrate, n- and iso-valerate, and n- and iso-caproate (total) <0.2%. Further components detectable included alanine, glycine, choline, phosphorylcholine, trimethylamine oxide, methanol, glycolate and assorted saccharides. In view of their high dissociation constants (K(a)), our results demonstrate that formic and pyruvic acids (K(a) = 1.77 x 10(-4) and 3.20 x 10(-3) mol/dm(3), respectively) contribute substantially to the decreased pH values associated with active caries lesions (cf. lactate K(a) = 1.40 x 10(-4) mol/dm(3)), and hence the pathogenesis of primary root caries.

Multicomponent evaluations of the oxidising actions and status of a peroxoborate-containing tooth-whitening system in whole human saliva using high resolution proton NMR spectroscopy.

High resolution 1H nuclear magnetic resonance (NMR) spectroscopy was employed to conduct a multicomponent investigation of the oxidation of salivary biomolecules by peroxoborate present in a tooth-whitening dentifrice formulation. The results acquired demonstrated that peroxoborate gave rise to the oxidative decarboxylation of the hydrogen peroxide scavenger pyruvate, a reaction generating acetate and CO2 as products. Experiments performed on chemical model systems confirmed the oxidative consumption of pyruvate by dentifrice-derived peroxoborate, and also revealed that the salivary electron donors cysteine and methionine (precursors to volatile sulphur compounds), were oxidised to cystine and methionine sulphoxide respectively. The biochemical and periodontal significance of these results is discussed.

In vivo absorption, metabolism, and urinary excretion of alpha,beta-unsaturated aldehydes in experimental animals. Relevance to the development of cardiovascular diseases by the dietary ingestion of thermally stressed polyunsaturate-rich culinary oils.

Thermal stressing of polyunsaturated fatty acid (PUFA)- rich culinary oils according to routine frying or cooking practices generates high levels of cytotoxic aldehydic products (predominantly trans-2-alkenals, trans,trans-alka-2,4-dienals, cis,trans-alka-2, 4-dienals, and n-alkanals), species arising from the fragmentation of conjugated hydroperoxydiene precursors. In this investigation we demonstrate that typical trans-2-alkenal compounds known to be produced from the thermally induced autoxidation of PUFAs are readily absorbed from the gut into the systemic circulation in vivo, metabolized (primarily via the addition of glutathione across their electrophilic carbon-carbon double bonds), and excreted in the urine as C-3 mercapturate conjugates in rats. Since such aldehydic products are damaging to human health, the results obtained from our investigations indicate that the dietary ingestion of thermally, autoxidatively stressed PUFA-rich culinary oils promotes the induction, development, and progression of cardiovascular diseases.

Multicomponent spectroscopic investigations of salivary antioxidant consumption by an oral rinse preparation containing the stable free radical species chlorine dioxide (ClO2.).

A multicomponent evaluation of the oxidative consumption of salivary biomolecules by a commercially-available oral rinse preparation containing an admixture of the stable free radical species chlorine dioxide (ClO2.) with chlorite anion (ClO2-) has been investigated using high resolution 1H NMR spectroscopy. The results obtained demonstrated that ClO2. and/or ClO2- present in this preparation effected the oxidative decarboxylation of salivary pyruvate (to acetate and CO2). Experiments conducted on chemical model systems confirmed the oxidative decarboxylation of pyruvate by this oral rinse, and also demonstrated that urate, thiocyanate anion, and the amino acids cysteine and methionine (precursors to volatile sulphur compounds responsible for oral malodour), were oxidatively consumed. The biochemical, periodontal and therapeutic significance of the results are discussed.

Molecular mechanisms of the bleaching actions associated with commercially-available whitening oral health care products.

An increased public awareness in dental aesthetics has resulted in the wide availability of techniques of tooth bleaching, both in the dental chair and at home. This article reviews the aetiology of tooth discolouration both at the clinical and the molecular level, together with methods of alleviating such discolouration. Much of the therapeutic and aesthetic actions of commercially-available tooth whiteners, gels, oral rinses and other dentifrices are predominantly dependent on their ability to act as oxidants. A novel method of evaluating these aspects of dentifrice activity is also described: high resolution proton nuclear magnetic resonance (NMR) spectroscopy is a virtually non-invasive, multi component bioanalytical technique that can be employed to study oxidation/reduction reactions at the molecular level and is utilised here to investigate the mechanisms of action of a newly developed dentifrice (Ultrawhite Opal, Janina International). Such methodology also offers much potential for studies concerning the numerous chemical reactions occurring within the oral environment.