Regulation of heptaspanning-membrane-receptor function by dimerization and clustering

G-protein-coupled receptors form homomers and heteromers; agonist-induced conformational changes within interacting receptors of the oligomer modify their pharmacology, signalling and/or trafficking. When these receptors are activated, the oligomers rearrange and cluster and a novel mechanism of receptor-operation regulation by oligomer intercommunication is possible. This intercommunication would be assisted by components of the plasma membrane and by scaffolding proteins. Receptor cross-sensitization, cross-desensitization and novel, integrated receptor responses can then develop between oligomeric receptor complexes of the cluster without direct contact between them. This concept gives a new perspective to the understanding of neurotransmission and neuronal plasticity.     

Metabotropic glutamate type 1alpha receptor localizes in low-density caveolin-rich plasma membrane fractions

Recent evidence suggest that many G protein-coupled receptors (GPCR) and signalling molecules localize in microdomains of the plasma membrane. In this study, flotation gradient analysis in the absence of detergents demonstrated the presence of the metabotropic glutamate receptor type 1alpha (mGlu1alpha) in low-density caveolin-enriched membrane fractions (CEMF) in permanently transfected BHK cells. BHK-1alpha cells exhibit a similar pattern of staining for caveolin-1 and caveolin-2, and these two proteins show a high degree of co-localization with mGlu1alpha receptor as demonstrated by immunogold and confocal laser microscopy. The presence of mGlu1alpha in CEMF was also demonstrated by co-immunoprecipitation of mGlu1alpha receptor using antibodies against caveolin proteins. Activation of the mGlu1alpha receptor by agonist increased extracellular signal-regulated kinases phosphorylation in CEMF and not in high-density membrane fractions (HDMF), suggesting that mGlu1alpha receptor-mediated signal transduction could occur in caveolae-like domains. Overall, these results clearly show a molecular and functional association of mGlu1alpha receptor with caveolins.     

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.     

Ligand-induced caveolae-mediated internalization of A1 adenosine receptors: morphological evidence of endosomal sorting and receptor recycling

The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the receptor sorting and recycling was studied in the smooth muscle cell line DDT(1)MF-2, by binding assays, by confocal microscopy, and at the structural level. The use of cholera toxin-binding subunit adsorbed to gold as a specific probe for labeling the ganglioside GM(1) and immunoelectron microscopy techniques showed that agonist stimulation produced a clustering and sequestration of adenosine receptors in caveolae. Furthermore, pull-down experiments showed there to be a direct interaction between the C-terminal domain of A(1)R and caveolin-1. Addition of exogenous adenosine deaminase (ADA), a protein that binds to A(1)R and acts as a receptor activity modifying protein (RAMP) stimulated R-PIA-induced A(1) receptor internalization. Finally, the sorting and recycling of A(1)R/ADA complexes was analyzed. Detailed electron microscopy revealed that A(1)R/ADA complexes internalize together through caveolae, are differentially sorted in endosomes, and are recycled back to the cell surface by different groups of recycling endosomes. These results give insight into the spatiotemporal regulation and traffic of A(1)R and RAMPs.     

Fungal fragments as indoor air biocontaminants

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.     

Assessing bactericidal properties of materials: the case of metallic surfaces in contact with air

A new method for assessing bactericidal properties of metallic materials, soiled by aerosol, was developed and applied to stainless steel in conditions close to reality. The airborne bacteria survival on different stainless steel grades and massive copper is presented here. The investigating bacterium was Enterococcus faecalis, which is a well-known contaminant strain in the indoor environments. It was observed that the bacterial aerosol lethality increased proportionally with the relative humidity (RH) of the environment. A significant difference in survival rate was measured depending on the tested supports, the greatest lethality being observed on clean massive copper. Moreover, the addition of nutrients on metallic surfaces, even in small quantities, was enough to ensure the revival of quiescent microorganisms.     

FFA cause hepatic insulin resistance by inhibiting insulin suppression of glycogenolysis

Free fatty acids (FFA) have been shown to inhibit insulin suppression of endogenous glucose production (EGP). To determine whether this is the result of stimulation by FFA of gluconeogenesis (GNG) or glycogenolysis (GL) or a combination of both, we have determined rates of GNG and GL (with (2)H(2)O) and EGP in 16 healthy nondiabetic volunteers (11 males, 5 females) during euglycemic-hyperinsulinemic (~450 pM) clamping performed either with or without simultaneous intravenous infusion of lipid plus heparin. During insulin infusion, FFA decreased from 571 to 30 micromol/l (P < 0.001), EGP from 15.7 to 2.0 micromol x kg(-1) x min(-1) (P < 0.01), GNG from 8.2 to 3.7 micromol x kg(-1). min(-1) (P < 0.05), and GL from 7.4 to -1.7 micromol x kg(-1). min(-1) (P < 0.02). During insulin plus lipid/heparin infusion, FFA increased from 499 to 1,247 micromol/l (P < 0.001). EGP decreased 64% less than during insulin alone (-5.1 +/- 0.7 vs. -13.7 +/- 3.4 micromol x kg(-1). min(-1)). The decrease in GNG was not significantly different from the decrease of GNG during insulin alone (-2.6 vs. -4.5 micromol x kg(-1). min(-1), not significant). In contrast, GL decreased 66% less than during insulin alone (-3.1 vs. -9.2 micromol x kg(-1). min(-1), P < 0.05). We conclude that insulin suppressed EGP by inhibiting GL more than GNG and that elevated plasma FFA levels attenuated the suppression of EGP by interfering with insulin suppression of GL.     

Laser-based micromanipulation for separation and identification of individual Frankia vesicles

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.     

Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.