Detection of cathepsin S cysteine protease in human brain tumour microdialysates in vivo.

Microdialysis enables the chemistry of extracellular ?uid in body tissues to be measured. Extracellular proteases such as the cysteine protease, cathepsin S (CatS), are thought to facilitate astrocytoma invasion. Microdialysates obtained from human brain tumoursin vivo were subjected to cathepsin S activity and ELISA assays. Cathepsin S ELISA expression was detected in ?ve out of 10 tumour microdialysates, while activity was detected in ?ve out of 11 tumour microdialysates. Cathepsin S expression was also detected in microdialysate from the normal brain control although no activity was found in the same sample. While some re?nements to the technique are necessary, the authors demonstrate the feasibility and safety of microdialysis in human astrocytomasin vivo. Characterisation of the extracellular environment of brain tumoursin vivo using microdialysis may be a useful tool to identify the protease pro?le of brain tumours.

Antimicrobial activity of truncated alpha-defensin (human neutrophil peptide (HNP)-1) analogues without disulphide bridges.

Antimicrobial peptides play an important role in host defence, particularly in the oral cavity where there is constant challenge by microorganisms. The a-defensin antimicrobial peptides comprise 30–50% of the total protein in the azurophilic granules of human neutrophils, the most abundant of which is human neutrophil peptide 1 (HNP-1). Despite its antimicrobial activity, a limiting factor in the potential therapeutic use of HNP-1 is its chemical synthesis with the correct disulphide topology. In the present study, we synthesised a range of truncated defensin analogues lacking disulphide bridges. All the analogues were modelled on the C-terminal region of HNP-1 and their antimicrobial activity was tested against a range of microorganisms, including oral pathogens. Although there was variability in the antimicrobial activity of the truncated analogues synthesised, a truncated peptide named 2Abz23S29 displayed a broad spectrum of antibacterial activity, effectively killing all the bacterial strains tested. The finding that truncated peptides, modelled on the C-terminal ß-hairpin region of HNP-1 but lacking disulphide bridges, display antimicrobial activity could aid their potential use in therapeutic interventions.

Auditory sensory memory and working memory processes in children with normal hearing and cochlear implants.

There can be wide variation in the level of oral/aural language ability that prelingually hearing-impaired children develop after cochlear implantation. Automatic perceptual processing mechanisms have come under increasing scrutiny in attempts to explain this variation. Using mismatch negativity methods, this study explored associations between auditory sensory memory mechanisms and verbal working memory function in children with cochlear implants and a group of hearing controls of similar age. Whilst clear relationships were observed in the hearing children between mismatch activation and working memory measures, this association appeared to be disrupted in the implant children. These findings would fit with the proposal that early auditory deprivation and a degraded auditory signal can cause changes in the processes underpinning the development of oral/aural language skills in prelingually hearing-impaired children with cochlear implants and thus alter their developmental trajectory

Fractal rotation isolates mechanisms for form-dependent motion in human vision

Here, we describe a motion stimulus in which the quality of rotation is fractal. This makes its motion unavailable to the translationbased motion analysis known to underlie much of our motion perception. In contrast, normal rotation can be extracted through the aggregation of the outputs of translational mechanisms. Neural adaptation of these translation-based motion mechanisms is thought to drive the motion after-effect, a phenomenon in which prolonged viewing of motion in one direction leads to a percept of motion in the opposite direction. We measured the motion after-effects induced in static and moving stimuli by fractal rotation. The after-effects found were an order of magnitude smaller than those elicited by normal rotation. Our findings suggest that the analysis of fractal rotation involves different neural processes than those for standard translational motion. Given that the percept of motion elicited by fractal rotation is a clear example of motion derived from form analysis, we propose that the extraction of fractal rotation may reflect the operation of a general mechanism for inferring motion from changes in form.

Linoleic acid increases monocyte chemotaxis and adhesion to human aortic endothelial cells through protein kinase C- and cyclooxygenase-2-dependent mechanisms

The effects of polyunsaturated n-6 linoleic acid on monocyte-endothelial interactions were investigated with particular emphasis on the expression of platelet/endothelial cell adhesion molecule (PECAM)-1 and the role of protein kinase C (PKC) and cyclooxygenase-2 (COX-2). As a diet rich in polyunsaturated fatty acids may favour atherosclerosis in hyperglycaemia, this study was performed in both normal and high-glucose media using human aortic endothelial cells (HAEC). The HAEC were preincubated with normal (5 mM) or high (25 mM) d-glucose for 3 days before addition of fatty acids (0.2 mM) for 3 days. Linoleic acid enhanced PECAM-1 expression independently of tumor necrosis factor (TNF)-a and significantly increased TNF-a-induced monocyte adhesion to HAEC in comparison to the monounsaturated n-9 oleic acid. Chronic glucose treatment (25 mM, 6 days) did not modify the TNF-a-induced or fatty acid-induced changes in monocyte binding. The increase in monocyte binding was accompanied by a significant increase in E-selectin and vascular cell adhesion molecule (VCAM)-1 expression and could be abrogated by an interleukin (IL)-8 neutralising antibody and by the PKC and COX inhibitors. Inhibition of PKC-d reduced VCAM-1 expression regardless of experimental condition and was accompanied by a significant decrease in monocyte binding. Conditioned medium from linoleic acid-treated HAEC grown in normal glucose conditions significantly increased THP-1 chemotaxis. These results suggest that linoleic acid-induced changes in monocyte chemotaxis and subsequent binding are not solely mediated by changes in adhesion molecule expression but may be due to secreted factors such as IL-8, monocyte chemoattractant protein-1 or prostaglandins (PGs) such as PGE2, as IL-8 neutralisation and COX-2 inhibition reduced monocyte binding without changes in adhesion molecule expression.

Potential roles for the PIM1 kinase in human cancer - A molecular and therapeutic appraisal

In vitro experiments have shown the PIM1 kinase to have diverse biological roles in cell survival, proliferation and differentiation. In humans, PIM1 is often expressed in both normal and transformed cells. The PIM1 kinase is a true oncogene implicated in early transformation and tumour progression in haematopoietic malignancies and prostate carcinomas. it is associated with aggressive subgroups of lymphoma, is a marker of poor prognosis in prostate carcinomas and has been suggested to have a role in hormone insensitivity of prostate malignancies. PIM1 has a possible role in other carcinomas with 6p21 genomic alterations. On one hand, PIM1 (due to its role in malignancy) appears to be a promising target for drug development programmes but, on the other hand, the complexity of its molecular structure has posed challenges in the development of PIM1 inhibitors. In this review we discuss PIM1 expression in human tissues (including some new data from our laboratory), its role in human malignancies, as well as the possibilities and challenges in the development of target therapy for PIM1. (c) 2008 Elsevier Ltd. All rights reserved.

RUNX3 protein is overexpressed in human basal cell carcinomas

Basal cell carcinomas (BCC), which are the most common form of skin malignancy, are invariably associated with the deregulation of the Sonic Hedgehog (Shh) signalling pathway. As such, BCC represent a unique model for the study of interactions of the Shh pathway with other genes and pathways. We constructed a tissue microarray (TMA) of 75 paired BCC and normal skin and analysed the expression of beta-catenin and RUNX3, nuclear effectors of the wingless-Int (Wnt) and bone morphogenetic protein/transforming growth factor-beta pathways, respectively. In line with previous reports, we observed varying subcellular expression pattern of beta-catenin in BCC, with 31 cases (41%) showing nuclear accumulation. In contrast, all the BCC cases tested by the TMA showed RUNX3 protein uniformly overexpressed in the nuclei of the cancer cells. Analysis by Western blotting and DNA sequencing indicates that the overexpressed protein is normal and full-length, containing no mutation in the coding region, implicating RUNX3 as an oncogene in certain human cancers. Our results indicate that although the deregulation of Wnt signalling could contribute to the pathogenesis of a subset of BCC, RUNX3 appears to be a universal downstream mediator of a constitutively active Shh pathway in BCC.

Rational Design of Acridine-Based Ligands with Selectivity for Human Telomeric Quadruplexes

Structure-based modeling methods have been used to design a series of disubstituted triazole-linked acridine compounds with selectivity for human telomeric quadruplex DNAs. A focused library of these compounds was prepared using click chemistry and the selectivity concept was validated against two promoter quadruplexes from the c-kit gene with known molecular structures, as well as with duplex DNA using a FRET-based melting method. Lead compounds were found to have reduced effects on the thermal stability of the c-kit quadruplexes and duplex DNA structures. These effects were further explored with a series of competition experiments, which confirmed that binding to duplex DNA is very low even at high duplex:telomeric quadruplex ratios. Selectivity to the c-kit quadruplexes is more complex, with some evidence of their stabilization at increasing excess over human telomeric quadruplex DNA. Selectivity is a result of the dimensions of the triazole-acridine compounds; and in particular the separation of the two alkyl-amino terminal groups. Both lead compounds also have selective inhibitory effects on the proliferation of cancer cell lines compared to a normal cell line, and one has been shown to inhibit the activity of the telomerase enzyme, which is selectively expressed in tumor cells, where it plays a role in maintaining telomere integrity and cellular immortalization.

Glucose lowers the threshold for human aortic vascular smooth muscle cell migration: inhibition by protein phosphatase-2A

AIMS/HYPOTHESIS: Atherosclerosis, which occurs prematurely in individuals with diabetes, incorporates vascular smooth muscle cell (VSMC) chemotaxis. Glucose, through protein kinase C-beta(II) signalling, increases chemotaxis to low concentrations of platelet-derived growth factor (PDGF)-BB. In VSMC, a biphasic response in PDGF-beta receptor (PDGF-betaR) level occurs as PDGF-BB concentrations increase. The purpose of this study was to determine whether increased concentrations of PDGF-BB and raised glucose level had a modulatory effect on the mitogen-activated protein kinase/extracellular-regulated protein kinase pathway, control of PDGF-betaR level and chemotaxis.

METHODS: Cultured aortic VSMC, exposed to normal glucose (NG) (5 mmol/l) or high glucose (HG) (25 mmol/l) in the presence of PDGF-BB, were assessed for migration (chemotaxis chamber) or else extracted and immunoblotted.

RESULTS: At concentrations of PDGF-BB <540 pmol/l, HG caused an increase in the level of PDGF-betaR in VSMC (immunoblotting) versus NG, an effect that was abrogated by inhibition of aldose reductase or protein kinase C-beta(II). At higher concentrations of PDGF-BB (>540 pmol/l) in HG, receptor level was reduced but in the presence of aldose reductase or protein kinase C-beta(II) inhibitors the receptor levels increased. It is known that phosphatases may be activated at high concentrations of growth factors. At high concentrations of PDGF-BB, the protein phosphatase (PP)2A inhibitor, endothall, caused an increase in PDGF-betaR levels and a loss of biphasicity in receptor levels in HG. At higher concentrations of PDGF-BB in HG, the chemoattractant effect of PDGF-BB was lost (chemotaxis chamber). Under these conditions inhibition of PP2A was associated with a restoration of chemotaxis to high concentrations of PDGF-BB.

CONCLUSION/INTERPRETATION: The biphasic response in PDGF-betaR level and in chemotaxis to PDGF-BB in HG is due to PP2A activation.

Estrogen Receptor-beta Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ER beta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ER alpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that FRO expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17 beta-estradiol led to an ER beta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ER beta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ER beta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598-604]

Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2 alpha gain-of-function mutation

The hypoxia-inducible factors (HIFs; isoforms HIF-1 alpha, HIF-2 alpha, HIF-3 alpha) mediate many responses to hypoxia. Their regulation is principally by oxygen-dependent degradation, which is initiated by hydroxylation of specific proline residues followed by binding of von Hippel-Lindau (VHL) protein. Chuvash polycythemia is a disorder with elevated HIF. It arises through germline homozygosity for hypomorphic VHL alleles and has a phenotype of hematological, cardiopulmonary, and metabolic abnormalities. This study explores the phenotype of two other HIF pathway diseases: classic VHL disease and HIF-2 alpha gain-of-function mutation. No cardiopulmonary abnormalities were detected in classic VHL disease. HIF-2 alpha gain-of-function mutations were associated with pulmonary hypertension, increased cardiac output, increased heart rate, and increased pulmonary ventilation relative to metabolism. Comparison of the HIF-2 alpha gain-of-function responses with data from studies of Chuvash polycythemia suggested that other aspects of the Chuvash phenotype were diminished or absent. In classic VHL disease, patients are germline heterozygous for mutations in VHL, and the present results suggest that a single wild-type allele for VHL is sufficient to maintain normal cardiopulmonary function. The HIF-2 alpha gain-of-function phenotype may be more limited than the Chuvash phenotype either because HIF-1 alpha is not elevated in the former condition, or because other HIF-independent functions of VHL are perturbed in Chuvash polycythemia.-Formenti, F., Beer, P. A., Croft, Q. P. P., Dorrington, K. L., Gale, D. P., Lappin, T. R. J., Lucas, G. S., Maher, E. R., Maxwell, P. H., McMullin, M. F., O'Connor, D. F., Percy, M. J., Pugh, C. W., Ratcliffe, P. J., Smith, T. G., Talbot, N. P., Robbins, P. A. Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2 alpha gain-of-function mutation. FASEB J. 25, 2001-2011 (2011). www.fasebj.org

A comparison of four extraction methods for substance P, neurokinin A and calcitonin gene-related peptide from human dental pulp tissue

Measuring neuropeptides in biological tissues by radioimmunoassay requires efficient extraction that maintains their immunoreactivity. Many different methods for extraction have been described, but there is little information on optimal extraction methods for individual neuropeptides from human dental pulp tissue. The aim was therefore to identify an effective extraction procedure for three pulpal neuropeptides: substance P. neurokinin A and calcitonin gene-related peptide. Tissue was obtained from 20 pulps taken from teeth freshly extracted for orthodontic reasons. The pulp samples were divided into four equal groups and different extraction methods were used for each group. Boiling whole pulp in acetic acid gave the highest overall yield and, in addition, offered an easy and rapid means of pulp tissue processing. The use of protease inhibitors did not increase the recovery of the immunoreactive neuropeptides but did provide the best combination of maximal recoveries and minimal variability. These results should be useful for planning the extraction of these neuropeptides from human pulp tissue in future studies. (C) 1999 Elsevier Science Ltd. All rights reserved.

Acetylation of Rb by PCAF is required for nuclear localization and keratinocyte differentiation

Although the retinoblastoma protein (Rb) functions as a checkpoint in the cell cycle, it also regulates differentiation. It has recently been shown that Rb is acetylated during differentiation; however, the role of this modification has not been identified. Depletion of Rb levels with short hairpin RNA resulted in inhibition of human keratinocyte differentiation, delayed cell cycle exit and allowed cell cycle re-entry. Restoration of Rb levels rescued defects in differentiation and cell cycle exit and re-entry; however, re-expression of Rb with the major acetylation sites mutated did not. During keratinocyte differentiation, acetylation of Rb is mediated by PCAF and it is further shown that PCAF acetyltransferase activity is also required for normal differentiation. The major acetylation sites in Rb are located within the nuclear localization sequence and, although mutation did not alter Rb localization in cycling cells, the mutant is mislocalized to the cytoplasm during differentiation. Studies indicate that acetylation is a mechanism for controlling Rb localization in human keratinocytes, with either reduction of the PCAF or exogenous expression of the deacetylase SIRT1, resulting in mislocalization of Rb. These findings identify PCAF-mediated acetylation of Rb as an event required to retain Rb within the nucleus during keratinocyte differentiation.