The cDNA cloning and immunological characterization of hamster p53.

We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75% homologous to human and mouse p53, respectively. Immunoprecipitations of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.     

Rainbow trout p53: cDNA cloning and biochemical characterization.

We have cloned and sequenced the p53-encoding cDNA of rainbow trout (Salmo gairdneri). The encoded product contains the characteristics found in all p53 proteins: (i) the five highly conserved domains, (ii) an acidic N terminus, (iii) a hydrophilic C terminus, and (iv) a penultimate serine residue. Furthermore, we demonstrate that the rainbow trout p53 is able to specifically interact with the SV40 large T antigen.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation.

In an effort to correlate the biological activity of the p53 protein with its conformation, we analysed 14 p53 mutants representative of the most frequently observed protein alterations in human cancers, at codons 175, 248 and 273 (22% of all mutations thus far reported), all three of which contained a CpG dinucleotide. Strikingly, most of the mutants at codons 248 and 273 did not display any change in their conformation, as probed by monoclonal antibodies PAb240 and PAb1620 or by binding to hsp70 protein. For all 14 mutants tested, we found a strict correlation between the transactivation properties of p53, tested either on RGC sequences or using the WAF-1 promoter, and inhibition of cell proliferation. All these mutants showed nuclear localization. Several mutants, present at a low incidence in human tumours, displayed wild-type activity in all our assays, suggesting that the presence of a mutation is not strictly correlated with p53 protein inactivation in tumours. Further analysis of nine thus far undescribed p53 mutants at codon 175 revealed a wild-type or mutant behaviour. All these results suggest that the occurrence of a mutation is dependent on two criteria: (i) the mutability of a given codon, such as those containing a CpG dinucleotide; (ii) the resulting amino acids, eventually leading to synthesis of a p53 conferring a growth advantage on the cell.     

The influence of cathelicidin LL37 in human anti-neutrophils cytoplasmic antibody (ANCA)-associated vasculitis

Introduction

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterised by the autoinflammation and necrosis of blood vessel walls. The renal involvement is commonly characterised by a pauci-immune crescentic glomerulonephritis (PiCGN) with a very rapid decline in renal function. Cathelicidin LL37, an endogenous antimicrobial peptide, has recently been implicated in the pathogenesis of autoimmune diseases. To assess whether serum LL37 reflects renal crescentic formation, we measured the serum levels of LL37 in AAV patients with and without crescentic glomerulonephritis (crescentic GN) as compared to healthy controls (HCs). We also analysed the correlation of the serum levels of LL37 and interferon-α (IFN-α) with the clinical characteristics of the patients.

Methods

The study population consisted of 85 AAV patients and 51 HCs. In 40 ANCA-positive patients, a parallel analysis was performed, including the assessment of LL37 and IFN-α levels in the serum and renal biopsies. Of those studied, 15 AAV patients had biopsy-proven crescentic GN, and 25 AAV patients lacked crescent formation. The serum levels of cathelicidin LL37 and IFN-α were both measured by ELISA, and the clinical and serological parameters were assessed according to routine procedures. Immunofluorescence staining was performed on frozen sections of kidney needle biopsies from AAV patients with crescentic GN.

Results

The serum levels of LL37 and IFN-α were significantly increased in AAV patients with crescentic GN compared to AAV patients without crescentic formation and HCs, and patients with high LL37 and IFN-α levels were more likely to be in the crescentic GN group. The LL37 levels were positively correlated with the IFN-α levels, and both LL37 and IFN-α levels showed a positive correlation with serum creatinine and no correlation with complement C3. The renal tissue of crescentic GN patients showed expression of LL37 and IFN-α at the Bowman’s capsule and extracellular sites, suggesting the active release of LL37 and IFN-α.

Conclusions

Significantly higher levels of LL-37 and IFN-α were observed in AAV patients, particularly those with crescentic formation, and LL37 and IFN-α were expressed in the renal tissue of patients with crescentic GN. These data suggest that serum levels of LL37 and IFN-α may reflect both local renal inflammation and systemic inflammation.     

Associations of rs1883832 and rs4810485 polymorphisms of CD40 gene with myocardial infarction in the Tunisian population

Context: Cluster of differentiation 40 (CD40), and its ligand CD40L, are major co-stimulatory molecules whose interactions are important in both cellular and humoral immunity, and has been suggested to play a role in the pathogenesis of acute coronary syndrome.

Objective: The aim of this study was to examine the association of CD40 polymorphisms (-1?C>T (rs1883832) and 945G>T (rs4810485)) and myocardial infarction (MI), and to test the association of CD40 gene haplotypes with MI in Tunisians.

Materials and methods: Three hundred and fifty MI patients and 301 apparently healthy controls were included in the study. The polymorphisms of CD40 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Results: There were significant differences in the genotype and allele frequencies of CD40 gene -1?C>T (rs1883832) polymorphism between cases and controls. Stratifying according to gender, the association between the TT genotype and MI was statistically significant in males, only. Haplotype analysis revealed that the C-T and T-G haplotypes were associated with an increased risk of MI (p?=?0.012 and p?<?0.001, respectively).

Conclusions: Our work showed a significant association between the -1?C>T (rs1883832) polymorphism of the CD40 gene and MI in the Tunisians.     

Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release

Human polymorphonuclear neutrophils (PMNs) express surface receptors for various inflammatory mediators, including IgE and IL-4. Recently, the IL-9R locus has been genetically linked to asthma and bronchial hyperresponsiveness in humans. In this study, we evaluated expression of the IL-9R and the effect of IL-9 on human PMNs. RT-PCR analysis showed the presence of IL-9Ralpha-chain mRNA in PMN RNA preparations from asthmatic patients. Using FACS analysis, surface expression of IL-9Ralpha was detected on PMNs freshly isolated from asthmatics, and to a lesser extent on normal controls. In addition, protein expression of IL-9Ralpha was also detected in peripheral blood and bronchoalveolar lavage PMNs. Furthermore, functional studies showed that IL-9 stimulation of PMNs results in the release of IL-8 in a concentration-dependent manner. The anti-IL-9 neutralizing Ab suppressed this effect, but had no effect on GM-CSF-induced IL-8 release from PMNs. Taken together, these findings suggest a novel role for PMNs in allergic disease through the expression and activation of the IL-9R.     

Liquid chromatography “on-flow” 1H nuclear magnetic resonance on native glycosphingolipid mixtures together with gas chromatography/mass spectrometry on the released oligosaccharides for screening and characterisation of carbohydrate-based antigens from pig lungs

Glycosphingolipids were prepared from pig lung and pooled into two fractions with (i) 3 sugar residues, and (ii) 3 sugar residues. Oligosaccharides were prepared and used for gas chromatography, gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The glycolipid fractions i and ii were further characterised and purified using a novel method based on high performance liquid chromatography on-flow proton nuclear magnetic resonance. The LC on-flow NMR technique showed good chromatographic separation and gave NMR spectral information which could be used as guidance for pooling of the separated mixture glycolipids. Conventional ¹H NMR, thin layer immunostaining, gas chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry were used to characterise the glycolipids and to validate LC-NMR spectral data.     

Effects of cadmium and copper on peroxidase, NADH oxidase and IAA oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots

Twelve-day-old seedlings of pea (Pisum sativum L.) that were treated for 4 days by 20 and 100 micromol/l Cd(NO3)2 or CuSO4 showed a growth reduction in all organs. From root protein extracts, the activities of guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11), coniferyl alcohol peroxidase (CAPX), NADH oxidase, and indole-3-acetic acid (IAA) oxidase were measured in covalently--and ionically--[symbol: see text] bound cell wall, soluble, and microsomal membrane fractions. With the exception of 20 micromol/l Cu, metal treatments enhanced GPX activity in all fractions. Only IAA oxidase activity was metal-elevated in the covalently bound cell wall fraction, while the ionic one showed Cd stimulation for all assayed enzymic activities. These effects were not entirely observed in Cu-treated plants, since APX and IAA oxidase activities were only enhanced in this fraction. However, soluble extract showed stimulation of APX activity, while in the microsomal fraction metal exposure also increased the activities of CAPX and NADH oxidase. Differential responses of root cell fractions to the presence of cadmium and copper ions are discussed in regard to the contribution of their enzymic capacities in antioxidant, lignification, and auxin degradation pathways. Comparisons between metals and dose effects are also underlined.