Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.     

Synthesis,crystal structure and antibacterial activity of new highly functionalized ionic compounds based on the imidazole nucleus

Several new highly functionalized imidazolium derivatives were synthesized, via appropriate synthetic routes, using imidazole, 1-methylimidazole and 2-phenyl-1-methylimidazole as key intermediates. The antibacterial activity of the prepared compounds was evaluated against: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella thipymurium using disk-diffusion and MIC methods. Crystal X-ray structures are reported for six compounds.     

In vivo IL-12 and IL-8 production in hydatic patients and their possible diffusion in hydatid cysts

Cystic echinococcosis (CE) is caused by infection with the larval stage of the cestode Echinococcus granulosus. It is one of the world's major zoonotic infections. Variability and severity of clinical expression of this parasitosis are associated with duration and intensity of infection. They are also related to the variety of human immunological responses to the hydatic antigens. The aim of this work is to study the inflammatory response associated with human hydatidosis by evaluating the possible roles of the proinflammatory cytokines in hydatic patients. We investigated the patterns of IL-12 and IL-8 in serum from Algerian hydatic patients. Serum IL-12 and IL-8 levels are significantly higher in patients with hydatidosis than in control subjects. Furthermore, cytokines secretion correlates with disease statues (cystic localizations and clinical stage). These data indicate that infection with E. granulosus is associated with high levels of circulating IL-12 and IL-8. Moreover, our data, to our knowledge, constitute the first report of IL-12 and IL-8 diffusion into the hydatid cyst. Our results underline the permeability of the cyst wall to the soluble immune system of the host. The relationship between cyst fertility and cytokine infiltration indicates a strong host-parasite interaction. All these findings have important implications for the diagnosis of hydatidosis in humans.     

Evidence of Airborne Excretion of Pneumocystis carinii during Infection in Immunocompetent Rats. Lung Involvement and Antibody Response

To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.     

Protein sequence comparison based on the wavelet transform approach

A protein's chemical properties, the chain conformation, the function of the protein and its species specificity are determined by the information contained in the amino acid sequence. Proteins of similar functions have at some level sequential identical amino acid sequences. The closer the phylogenetic relationship, the more similar are the sequences. To find the similarities between two or more protein sequences is of great importance for protein sequence analysis. The differences in the amino acid sequences permit the construction of a family tree of evolution. In this work, a comparison method was devised that is capable of analysing a protein sequence 'hierarchically', i.e. it can examine a protein sequence at different spatial resolutions. Based on a wavelet decomposition of protein sequences and a cross-correlation study, a sequence-scale similarity concept is proposed for generating a similarity vector, which renders the comparison of two sequences feasible at different spatial resolutions (scales). This new similarity concept is an expansion of the conventional sequence similarity, which only takes into account the local pairwise amino acid match and ignores the information contained in coarser spatial resolutions.     

Increased IL-1β levels are associated with an imbalance of “oxidant/antioxidant” status during Behçet’s disease

Background

Behçet’s disease is a multisystem disease. It stands at the crossroad between the autoimmunity and auto-inflammatory disorders. In this study, we sought to address a relationship that might exist between interleukin-1β (IL-1β) and the oxidants/antioxidants markers in Behçet’s patients.

Methods

Behçet’s disease patients (n = 78: active stage, n = 28; inactive stage, n = 50) and 41 healthy controls have been included in our study. In this context, we investigated the plasma levels of IL-1β and the nitrosative/oxidative markers: nitric oxide (NO), advanced oxidative protein products (AOPP) and fatty acids peroxidation-malondialdehyde (MDA). The antioxidant system was assessed by measuring the plasma level of superoxide dismutase (SOD) activity. The Mann-Whitney’s U and Pearson’s correlation tests were used for statistical analyses.

Results

Our case-control study showed that patients in active stage displayed higher plasma levels of IL-1β, NO, AOPP and MDA versus healthy controls and patients in inactive stage. Patients in active stage showed significantly lower SOD levels related to patients in inactive stage and healthy controls respectively, whereas patients in inactive stage showed statistically insignificant SOD level versus healthy controls. Correlation studies showed a significant positive correlation between IL-1β and AOPP, IL-1β and NO, and negative correlation between IL-1β and SOD among Behçet’s disease patients. In addition, we showed positive correlation between AOPP and NO, AOPP and MDA and negative correlation between NO and SOD, AOPP and SOD in Behçet’s disease patients.

Conclusion

Interestingly, our study revealed that IL-1β levels increased and correlated with an imbalance of oxidants/antioxidants system, especially during active stage of Behçet disease. Collectively, our study indicates a possible link between IL-1β production and nitrosative/oxidative markers during Behçet’s disease. Exploiting this relationship might provide valuable outputs in the follow-up and prognosis of Behçet’s disease with a potential therapeutic value.

     

Epidermal growth factor receptor (EGFR) abundance correlates with p53 and Bcl-2 accumulation and patient age in a small cohort of North African nasopharyngeal carcinoma patients

In North Africa, nasopharyngeal carcinoma (NPC) is characterized by a bimodal distribution involving a juvenile (≤ 30 years old) and an elder population (> 30 years old). The Epstein Barr virus oncogene LMP1, the anti-apoptotic Bcl-2 protein and the tumor suppressor p53 have recently emerged as biomarkers of the disease. EGFR/ErbB1 expression is detected in the majority of NPC tumors with advanced disease. To obtain greater insight into the potential oncogenic mechanisms specific to these two NPC populations, we examined the correlation between EGFR expression and patient age, and determined the molecular profiles of its associations with the biomarkers of NPC. We performed an immunohistochemical analysis of the latter molecules in NPC specimens from eleven Algerian patients (six patients ≤ 30 years of age and five patients > 30 years of age) using the LSAB method. Evaluation of the biopsies, based on the intensity of staining and the percentage of positive cells, showed that LMP1 expression was higher in patients under 30 years of age. Conversely, EGFR, like Bcl-2 and p53, was significantly up-regulated in tumors from elderly patients. Analysis of all tumors showed that EGFR expression was constantly (100%) associated with high p53 nuclear accumulation and Bcl-2 expression in LMP1-positive tissues. Biopsies negative for Bcl-2 staining were found to display low amounts of p53 (100%), and to be constantly negative for EGFR (100%). Molecular classification of all NPC tissues showed that the majority of patients displaying a EGFR+/LMP1+/Bcl-2+/p53-high molecular pattern were in the older age group. On the other hand, the most of the EGFR negative results were associated with the juvenile form of the disease and were characterized by an important diversity of molecular patterns. Our preliminary results suggest that in Algerian patients, the bimodal distribution of NPC might be related to distinct expression profiles of viral and cellular biomarkers of NPC.