Intracellular cross-talk between the GPCR CXCR1 and CXCR2: role of carboxyl terminus phosphorylation sites

In the present study, we used the human chemokine receptors CXCR1 and CXCR2 as a model system for the study of intracellular cross-talk between two closely related G protein-coupled receptors (GPCR). In cells expressing either CXCR1 or CXCR2, exposure to the CXCL8 ligand resulted in prominent reduction in cell surface expression of the receptors. We have shown previously that the reduction in cell surface expression of CXCR1 and CXCR2, to be termed herein "down-regulation", is significantly lower in cells expressing both receptors together. Now we show that reduced receptor down-regulation was specific to the CXCR1+CXCR2 pair. Also, CXCR2 carboxyl terminus phosphorylation sites were required for inducing inhibition of CXCR1 down-regulation, and vice versa. Accordingly, phosphorylation of CXCR2 carboxyl terminus domain was intact when expressed together with CXCR1. Moreover, specific carboxyl terminus phosphorylation sites on each of the wild type receptors protected them from more severe inhibition of down-regulation, induced by joint expression with the other receptor. When concomitantly expressed, CXCR1 and CXCR2 were impaired in recycling to the plasma membrane, despite their undergoing intact dephosphorylation. Overall, we show that cross-talk between two GPCR is manifested by impairment of their intracellular trafficking, primarily of ligand-induced down-regulation, via carboxyl terminus phosphorylation sites.     

Polyphenol-induced dissociation of various amyloid fibrils results in a methionine-independent formation of ROS

Fibrillization of amyloid polypeptides is accompanied by formation of reactive oxygen species (ROS), which, in turn, is assumed to further promote amyloid-related pathologies. Different polyphenols, all of which are established antioxidants, cause dissociation of amyloid fibrils. This study addresses the latter, poorly understood process. Specifically, we have investigated the dissociation of Abeta(42) fibrils by six different polyphenols, using electron microscopy and spectrofluorometric analysis. Simultanously, we have monitored the production of ROS using electron spin resonance (ESR) and the commercially available peroxide assay kit. Using the same methods we found that curcumin, one of the most potent destabilizing agents of Abeta(42), induced dissociation of fibrils of other amyloid polypeptides [Abeta(40), Abeta(42)Nle35, islet amyloid polypeptide and a fragment of alpha-synuclein]. When the solution contained traces of transition metal, all the dissociation reactions were accompanied by ROS formation, independent of the presence of a methionine residue. Kinetic studies show that the formation of ROS lags behind dissociation, indicating that if casual relationship exists between these two processes, then ROS formation may be considered a consequence and not a cause of dissociation. These findings open new avenues in amyloid research that will be required to gain further understanding of our results and of their implications.     

Comparative proteomics of the developing fish (zebrafish and gilthead seabream) oocytes

The maturation process of fish oocytes involves both protein biosynthesis within the oocytes and uptake from the plasma. To follow the changes in the proteins repertoires of fish oocytes during maturation, we performed a large-scale proteomics analysis using one and two-dimensional electrophoresis, multi-dimensional protein identification technology (MudPIT) and tandem mass-spectrometry. A large number of proteins were identified and a map of the vitellogenin derived yolk proteins; lipovitellin and phosvitin, was established (the vitellogenin map), reflecting the posttranslational processing of the different vitellogenins gene-products and their accumulation. Such protein patterns are potentially useful for molecular staging and for quality-control of maturing oocytes. Furthermore, proteomics analyses of single oocytes were used to demonstrate molecular variability between morphologically similar oocytes of same or different fish specimens. Proteins of interest detected in this study include proteins that may serve as maternal factors, such as TCP1, serpin A1 and importin alpha1. The large similarity between the proteins repertoires of fish oocytes and other species, such as mammals and insects, demonstrate the evolutionary conservation of oocyte maturation across diverse species gap.     

Activation of local and systemic anti-tumor immune responses by ablation of solid tumors with intratumoral electrochemical or alpha radiation treatments

Cancer, the most devastating chronic disease affecting humankind, is treated primarily by surgery, chemotherapy, and radiation therapy. Surgery and radiotherapy are mainly used for debulking the primary tumor, while chemotherapy is the most efficient anti-metastatic treatment. To control better metastatic cancer, the host immune system should be stimulated. Yet, successful specific stimulation of the immune system against tumors was seldom achieved even in antigenic tumors. Our working hypothesis is that aggressive in situ tumor ablation can release tumor antigens and danger signals, which will enhance anti-tumor T cell responses resulting in the destruction of residual malignant cells in primary tumors and distant metastases. We developed two efficient in situ ablation treatments for solid cancer, which can be used to destroy the primary tumors and stimulate anti-tumor immune responses. The first treatment, electrochemical ablation, is applied through intratumoral electrodes, which deliver unipolar-pulsed electric currents. The second treatment, diffusing alpha-emitters radiation therapy (DaRT), is based on intratumoral ²²⁴Ra-loaded wire(s) that release by recoil its daughter atoms. These short-lived alpha-emitting atoms spread in the tumor and spray it with lethal alpha particles. It was confirmed that these treatments effectively destroy various malignant animal and human primary solid tumors. As a consequence of such tumor ablation, tumor-derived antigenic material was released and provoked systemic T cell-dependent anti-tumor immunological reactions. These reactions conferred protection against a secondary tumor challenge and destroyed remaining malignant cells in the primary tumor as well as in distant metastases. Such anti-tumor immune responses could be further amplified by the immune adjuvant, CpG. Electrochemical ablation or DaRT together with chemotherapy and immunostimulatory agents can serve as treatment protocols for solid metastatic tumors and can be applied instead of or in combination with surgery.     

Assimilation of polysaccharides and glucose by major bacterial groups in the Delaware Estuary

The contribution of major bacterial groups to the assimilation of extracellular polymeric substances (EPS) and glucose in the Delaware Estuary was assessed using microautoradiography and fluorescence in situ hybridization. Bacterial groups contributed to EPS and glucose assimilation in part according to their distribution in the estuary. Abundance of the phylogenetic groups explained 35% and 55% of the variation in EPS and glucose assimilation, respectively. Actinobacteria contributed 70% to glucose assimilation in freshwater, while Alphaproteobacteria assimilated 60% of this compound in saline water. In contrast, various bacterial groups dominated the assimilation of EPS. Actinobacteria and Betaproteobacteria contributed the most in the freshwater section, whereas Cytophaga-like bacteria and Alpha- and Gammaproteobacteria participated in EPS assimilation in the lower part of the estuary. In addition, we examined the fraction of bacteria in each group that assimilated glucose or EPS. Overall, the fraction of bacteria in all groups that assimilated glucose was higher than the fraction that assimilated EPS (15 to 30% versus 5 to 20%, respectively). We found no correlation between the relative abundance of a group in the estuary and the fraction of bacteria actively assimilating glucose or EPS; the more active groups were often less abundant. Our results imply that the bacterial community in the Delaware Estuary is not controlled solely by "bottom-up" factors such as dissolved organic matter.     

Electroendocytosis: exposure of cells to pulsed low electric fields enhances adsorption and uptake of macromolecules

This study demonstrates alteration of cell surface, leading to enhanced adsorption of macromolecules (bovine serum albumin (BSA), dextran, and DNA), after the exposure of cells to unipolar pulsed low electric fields (LEF). Modification of the adsorptive properties of the cell membrane also stems from the observation of LEF-induced cell-cell aggregation. Analysis of the adsorption isotherms of BSA-fluorescein isothiocyanate (FITC) to the surface of COS 5-7 cells reveals that the stimulated adsorption can be attributed to LEF-induced increase in the capacity of both specific and nonspecific binding. The enhanced adsorption was consequently followed by increased uptake. At 20 V/cm the maximal binding and subsequent uptake of BSA-FITC attached to specific sites are 6.5- and 7.4-fold higher than in controls, respectively. The nonspecific LEF-induced binding and uptake of BSA are 34- and 5.2-fold higher than in controls. LEF-enhanced adsorption is a temperature-independent process, whereas LEF-induced uptake is a temperature-dependent one that is abolished at 4 degrees C. The stimulation of adsorption and uptake is reversible, revealing similar decay kinetics at room temperature. It is suggested that electrophoretic segregation of charged components in the outer leaflet of the cell membrane is responsible for both enhanced adsorption and stimulated uptake via changes of the membrane elastic properties that enhance budding and fission processes.     

A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents

Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein–protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10⁴ pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner.     

MLPA Subtelomere Analysis in Tunisian Mentally Retarded Patients

Subtelomeric rearrangements significantly contribute to idiopathic mental retardation and result in several mental retardation syndromes; however, most subtelomeric defects lack a characteristic phenotype. Thirty patients with unexplained mental retardation, a normal R banded karyotype at the 550 band, and no clinically recognizable syndrome were screened by Multiplex ligation-dependent probe amplification (MLPA). Four anomalies were identified: deletion 17q, duplications (4q), and associated duplications 15q and Xq. This duplication was found in two sisters of the proband. Anomalies were unidentified by the conventional technique. The prevalence of subtelomeric imbalances in our cohort of moderate to severe mental retardation is around 13% and is consistent with the literature. The sensitivity of the MLPA technique was characterized on cytogenetically verified positive and negative controls. MLPA is a fast, reliable, and relatively inexpensive technique to detect subtelomeric rearrangement in comparison with the fluorescence in situ hybridization (FISH) technique.