Factors modulating the blood feeding behavior and the electrophysiological responses of labral apical chemoreceptors to adenine nucleotides in the mosquito Aedes aegypti (Culicidae)

The feeding of Aedes aegypti (L.) on blood is induced by the presence of phagostimulants: adenine nucleotides. Three chemoreceptive cells in the labral apical sensilla can distinguish the presence of adenine nucleotides depending on the other stimulus components. This work aims at correlating the sensory information arising from the labral apical sensilla with the feeding behavior in response to the same stimuli. The saline stimulating solution, containing adenine nucleotides, is modulated by changing one of the following components: salt concentration, buffer or pH. Cell 3 that responds to NaCl in a dose dependent manner seems to have another unique modality. The response of this cell is unaffected by ATP when the stimulating solution is NaCl buffered by NaHCO(3). It responds at a higher spike frequency to the presence of ATP in a NaCl solution without NaHCO(3). Thus in the presence of ATP Cell 3 detects whether the NaCl solution is buffered by NaHCO(3). Both the blood feeding response and the sensory information from Cell 2 (which responds at high spike frequencies to the presence of ATP) are modulated by pH in a similar way. Both responses present a bi-modal response, with a major peak at pH 4.0 and a moderate peak at the most alkaline pH value tested.     

A hierarchy of lipid constructs for the sperm plasma membrane

We have presented a series of lipid constructs as models of the sperm plasma membrane. We also isolated the plasma membrane from rabbit sperm cells and characterized the lipid composition. The behavior of these various membrane systems was evaluated using a vesicle leakage assay, in which surfactant (nonoxynol-9, N-9; or benzalkonium chloride, BZK) exposure induced membrane permeabilization. These studies shed light on the relative importance and significance of particular components present in the lipid constructs. In particular, a highly unsaturated phospholipid component characterized by an ether-linkage to position 1 of the glycerol backbone (as opposed to the more conventional ester linkage) as well as the presence of sulfogalactosyl ceramide were found to have an effect on the surfactant-induced leakage response. The presence of cholesterol had the greatest influence on membrane behavior. The construct series also demonstrated the ability of the surfactants studied to discriminate between different membrane systems. We found that N-9 displayed little sensitivity to membrane composition while BZK showed specific behavior with the various membrane systems.     

Integrated Microfluidics for Protein Modification Discovery

Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system''s potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.Protein post-translational modifications (PTMs)¹ vastly diversify eukaryotic proteomes and are integrated in essentially all cellular processes (1). Proteomic approaches, such as mass spectrometry (MS), have been instrumental in monitoring global molecular dynamics for research and clinical applications (2–5). However, even in this modern era, large-scale analyses of PTMs by MS is challenging because of the limited number of modified peptides derived from proteins that, by themselves, may not be abundant. Moreover, comprehensive PTM analysis by MS often requires significant amounts of biological material that may not be available. PTM analysis using protein arrays can overcome these limitations because of the equimolar amount of the arrayed proteins (6, 7). Large-scale protein arrays have been successfully integrated into PTM research (8, 9). However, this technology relies on pre-purified proteins that are arrayed on a surface and thus, incompatible with biochemically challenging proteins, let alone insoluble proteins. Moreover, the production of recombinant protein arrays is impractical in-house. Therefore, such arrays cannot be used fresh, and they are inherently limited to certain designs, protein compositions, and model organisms of high commercial value. To overcome the abovementioned limitations, we designed a modular integrated microfluidic platform for PTM analysis (IMPA).     

Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1alpha (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.     

A hepatocellular carcinoma cell line producing mature hepatitis B viral particles

Current in vitro models for hepatitis B virus (HBV) are based on human hepatoblastoma cell lines transfected with HBV genome. The objective of this work was to develop an in vitro, hepatocellular carcinoma (HCC)-based system supporting HBV full replication and producing mature viral particles. The FLC4 human HCC cell line was stably transfected with a plasmid carrying a head-to-tail dimer of the adwHBV genome. One of the clones, FLC4A10II, exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. Furthermore, the cells produced HBV particles that were detected by a cesium chloride density gradient performed on the culture medium. Analysis by Southern blot revealed that HBV DNA has integrated into the FLC4A10II cell genome. The presence of HBV in the FLC4A10II cells did not cause alterations in cell morphology and the cells continued to resemble mature hepatocytes. They do exhibit a high mitotic activity. The new HBV stably transfected cell line, FLC4A10II, can serve as an important tool for further exploration of HBV host-pathogen interaction, viral life cycle, and for assessing new antiviral agents.     

Hepatitis C virus core protein associates with detergent-resistant membranes distinct from classical plasma membrane rafts

A subpopulation of hepatitis C virus (HCV) core protein in cells harboring full-length HCV replicons is biochemically associated with detergent-resistant membranes (DRMs) in a manner similar to that of markers of classical lipid rafts. Core protein does not, however, colocalize in immunofluorescence studies with classical plasma membrane raft markers, such as caveolin-1 and the B subunit of cholera toxin, suggesting that core protein is bound to cytoplasmic raft microdomains distinct from caveolin-based rafts. Furthermore, while both the structural core protein and the nonstructural protein NS5A associate with membranes, they do not colocalize in the DRMs. Finally, the ability of core protein to localize to the DRMs did not require other elements of the HCV polyprotein. These results may have broad implications for the HCV life cycle and suggest that the HCV core may be a valuable probe for host cell biology.     

RNA interference for antiviral therapy

Silencing gene expression through a process known as RNA interference (RNAi) has been known in the plant world for many years. In recent years, knowledge of the prevalence of RNAi and the mechanism of gene silencing through RNAi has started to unfold. It is now believed that RNAi serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing RNAi have been found in various viral pathogens. During the past few years, it has been demonstrated that RNAi, induced by specifically designed double-stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. However, many key questions and obstacles in the translation of RNAi into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsRNA that induces the system. It is expected that for the specific examples in which the delivery issue could be circumvented or resolved, RNAi may hold promise for the development of gene-specific therapeutics.     

Interleukin-5 receptor subunit oligomerization and rearrangement revealed by fluorescence resonance energy transfer imaging

Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, alpha and betac. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5Ralpha and betac were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5Ralpha-CFP and betac-YFP was observed in response to IL-5, indicative of heteromeric receptor alpha-betac subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Ralpha. Substantial FRET signals with betac-CFP and betac-YFP co-expressed in the absence of IL-5Ralpha demonstrated that betac subunits exist as preformed homo-oligomers. IL-5 had no effect on this betac-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing betac-CFP, betac-YFP, and nontagged IL-5Ralpha led to further increase in FRET efficiency. Observation of preformed betac oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on betac assembly in the presence of nontagged IL-5Ralpha provide direct evidence that IL-5 can cause higher order rearrangements of betac homo-oligomers. These results suggest that IL-5 and perhaps other betac cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit alpha), followed by binding of the ligand-subunit alpha complex to, and consequent rearrangement of, a ground state form of betac oligomers.