Endocytosis of ligand-activated plasma membrane receptors has been shown to contribute to the regulation of their downstream signaling. beta-arrestins interact with the phosphorylated tail of activated receptors and act as scaffolds for the recruitment of adaptor proteins and clathrin, that constitute the machinery used for receptor endocytosis. Visual- and beta-arrestins have a two-lobe, immunoglobulin-like, beta-strand sandwich structure. The recent resolution of the crystal structure of VPS26, one of the retromer subunits, unexpectedly evidences an arrestin fold in this protein, which is otherwise unrelated to arrestins. From a functional point of view, VPS26 is involved in the retrograde transport of the mannose 6-P receptor from the endosomes to the trans-Golgi network. In addition to the group of genuine arrestins and Vps26, mammalian cells harbor a vast repertoire of proteins that are related to arrestins on the basis of their PFAM Nter and Cter arrestin- domains, which are named Arrestin Domain- Containing proteins (ADCs). The biological role of ADC proteins is still poorly understood. The three subfamilies have been merged into an arrestin-related protein clan.This paper provides an overall analysis of arrestin clan proteins. The structures and functions of members of the subfamilies are reviewed in mammals and model organisms such as Drosophila, Caenorhabditis, Saccharomyces and Dictyostelium.

When Drosophila adults are placed into an open field arena, they initially exhibit an elevated level of activity followed by a reduced stable level of spontaneous activity. We have found that the initial elevated component arises from the fly's interaction with the novel arena since: 1) the increased activity is independent of handling prior to placement within the arena, 2) the fly's elevated activity is proportional to the size of the arena, and 3) the decay in activity to spontaneous levels requires both visual and olfactory input. These data indicate that active exploration is the major component of elevated initial activity. There is a specific requirement for the kurtz non-visual arrestin in the nervous system for both the exploration stimulated by the novel arena and also mechanically stimulated activity. kurtz is not required for spontaneous activity; kurtz mutants display normal levels of spontaneous activity and average the same velocities as wild type controls. Inhibition of dopamine signaling has no effect on the elevated initial activity phase in either wild type or in krz1 mutants. Therefore, the exploratory phase of open field activity requires kurtz in the nervous system, but is independent of dopamine's stimulation of activity.

Heterotrimeric G(o) is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo. Here we demonstrate that G(o) signaling is required for the formation of associative memory. In Drosophila melanogaster, pertussis toxin (PTX) is a selective inhibitor of G(o) signaling. The postdevelopmental expression of PTX within mushroom body neurons robustly and reversibly inhibits associative learning. The effect of G(o) inhibition is distributed in both gamma- and alpha/beta-lobe mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory. PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G(o) defines a new signaling pathway required in mushroom body neurons for the formation of associative memory.

Nonvisual arrestins are a family of multifunctional adaptor molecules that regulate the activities of diverse families of receptors including G protein-coupled receptors, frizzled, and transforming growth factor-beta receptors. These activities indicate broad roles in both physiology and development for nonvisual arrestins. Drosophila melanogaster has a single nonvisual arrestin, kurtz, which is found at high levels within the adult olfactory receptor neurons (ORNs), suggesting a role for this gene in modulating olfactory sensitivity. Using heat-induced expression of a krz cDNA through development, we rescued krz(1) lethality. The resulting adults lacked detectable levels of krz in the olfactory system. The rescued krz(1) homozygotes have an incompletely penetrant antennal structural defect that was completely rescued by the neural expression of a krz cDNA. The krz(1) loss-of-function adults without visible antennal defects displayed diminished behavioral responsiveness to both aversive and attractive odors and also demonstrated reduced olfactory receptor potentials. Both the behavioral and electrophysiological phenotypes were rescued by the targeted expression of the krz cDNA within postdevelopmental ORNs. Thus, krz is required within the nervous system for antennal development and is required later in the ORNs for the maintenance of olfactory sensitivity in Drosophila. The reduced receptor potentials in krz(1) antenna indicate that nonvisual arrestins are required for the early odor-induced signaling events within the ORNs.

Recently, we have successfully developed a two-photon excitation fluorescence resonance energy transfer (TPE-FRET)-based homogeneous immunoassay using two-photon excitable small organic molecule as the energy donor. In the present work, the newly emerging TPE-FRET technique was extended to the determination of oligonucleotide. A new TPE molecule with favorable two-photon action cross section was synthesized [2-(2,5-bis(4-(dimethylamino)styryl)-1H-pyrrol-1-yl)acetic acid, abbreviated as TP-COOH], with the tagged reactive carboxyl group allowing facile conjugation with streptavidin (SA). Employing the TP-COOH molecule as energy donor and black hole quencher 1 (BHQ-1) as acceptor, a TPE-FRET-based homogeneous competitive hybridization model was constructed via a biotin-streptavidin bridge. Through the hybridization between a biotinylated single-stranded DNA (ssDNA) and a BHQ-1-linked ssDNA, and the subsequent capture of the as-formed hybrid by TP-COOH labeled SA, the donor fluorescence was quenched due to the FRET between TP-COOH and BHQ-1. Upon the competition between a target ssDNA and the quencher-linked ssDNA toward the biotinylated oligonucleotide, the donor fluorescence was recovered in a target-dependent manner. Good linearity was obtained with the target oligonucleotide ranging from 0.08 to 1.52 muM. The method was applied to spiked serum and urine samples with satisfying recoveries obtained. The results of this work verified the applicability of TPE-FRET technique in hybridization assay and confirmed the advantages of TPE-FRET in complicated matrix.

ABSTRACT: BACKGROUND: Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology and function as ligand-gated cation channels. Consequently, the involvement of cyclic nucleotides and G proteins in insect odor reception is controversial. Since the heterotrimeric Goalpha subunit is expressed in Drosophila olfactory receptor neurons, we reasoned that Go acts together with insect odorant receptor cation channels to mediate odor-induced physiological responses. RESULTS: To test whether Go dependent signaling is involved in mediating olfactory responses in Drosophila, we analyzed electroantennogram and single-sensillum recordings from flies that conditionally express pertussis toxin, a specific inhibitor of Go in Drosophila. Pertussis toxin expression in olfactory receptor neurons reversibly reduced the amplitude and hastened the termination of electroantennogram responses induced by ethyl acetate. The frequency of odor-induced spike firing from individual sensory neurons was also reduced by pertussis toxin. These results demonstrate that Go signaling is involved in increasing sensitivity of olfactory physiology in Drosophila. The effect of pertussis toxin was independent of odorant identity and intensity, indicating a generalized involvement of Go in olfactory reception. CONCLUSION: These results demonstrate that Go is required for maximal physiological responses to multiple odorants in Drosophila, and suggest that OR channel function and G-protein signaling are required for optimal physiological responses to odors.

BACKGROUND: Selectivity is paramount for the successful implementation of noninvasive spectroscopic sensing for the painless measurement of blood glucose concentrations in people with diabetes. Selectivity issues are explored for different multivariate calibration models based on noninvasive near-infrared spectra collected from an animal model. METHODS: Noninvasive near-infrared spectra are collected through a fiber-optic interface attached to a thin fold of skin on the back of an anesthetized laboratory rat while glucose levels are varied in a controlled manner. RESULTS AND DISCUSSION: Partial least-squares (PLS) calibration models are generated from noninvasive spectra collected during a single, 2-hour blood glucose transient. Calibration vectors are compared for optimized PLS calibration models created with correct and incorrect assignments of glucose concentrations to each noninvasive spectrum. Although both PLS models appear functional and seem capable of predicting glucose concentrations accurately during this transient, only the model generated from correct glucose assignments gives a credible calibration vector. When correct glucose assignments are used, the PLS calibration vector matches the corresponding net analyte signal calibration vector. No similarity in these calibration vectors is evident when incorrect glucose assignments are used. CONCLUSIONS: Glucose-specific spectral information is present within noninvasive near-infrared spectra collected from a rat model using a transmission geometry. Apparently functional, yet incorrect, calibration models can be generated, and the propensity to create such false PLS calibration models calls into question the validity of past reports. An analysis of calibration vectors can provide valuable insight into the chemical basis of selectivity for multivariate calibration models of complex systems.

Ovarian cancer is 1 of the most significant malignancies in the Western world, and the antiangiogenesis strategy has been postulated for prevention and treatment of ovarian cancers. Kaempferol is a natural flavonoid present in many fruits and vegetables. The antiangiogenesis potential of kaempferol and its underlying mechanisms were investigated in two ovarian cancer cell lines, OVCAR-3 and A2780/CP70. Kaempferol mildly inhibits cell viability but significantly reduces VEGF gene expression at mRNA and protein levels in both ovarian cancer cell lines. In chorioallantoic membranes of chicken embryos, kaempferol significantly inhibits OVCAR-3-induced angiogenesis and tumor growth. HIF-1alpha, a regulator of VEGF, is downregulated by kaempferol treatment in both ovarian cancer cell lines. Kaempferol also represses AKT phosphorylation dose dependently at 5 to 20 muM concentrations. ESRRA is a HIF-independent VEGF regulator, and it is also downregulated by kaempferol in a dose-dependent manner. Overall, this study demonstrated that kaempferol is low in cytotoxicity but inhibits angiogenesis and VEGF expression in human ovarian cancer cells through both HIF-dependent (Akt/HIF) and HIF-independent (ESRRA) pathways and deserves further studies for possible application in angio prevention and treatment of ovarian cancers.

In Drosophila, two related G-protein-coupled receptors are members of the corticotropin releasing factor (CRF) receptor subfamily. We have previously reported that one of these receptors, encoded by CG8422 is a functional receptor for a diuretic hormone, DH(44). Here, we report that the other CRF receptor subfamily member, encoded by CG12370, is also a receptor for the DH(44) neuropeptide. The lines of evidence to support this identification include increases in cAMP levels due to CG12370 receptor activation and the recruitment of beta-arrestin-GFP to the plasma membrane in response to DH(44) application. We compared these features of the receptors DH44-R2 (encoded by CG12370) and DH44-R1 (encoded by CG8422) and found fundamental differences in signaling, association with the arrestins, and peptide sensitivity. We found that the sensitivity of DH44-R2 to the DH(44) peptide is lower than that of DH44-R1, specifically an estimated EC(50) of 7.98E-07 moll(-1) for DH(44) by DH44-R2 to an EC(50) of 5.12E-09 moll(-1) by DH44-R1 and found that previous reports on the sensitivity of the tubule to DH(44) is in agreement with our measurements of DH44-R2 sensitivity. We employed a specific RNAi construct to selectively knock-down DH44-R2 expression and this led to heightened sensitivity to osmotic challenges. The functional characterization of this diuretic hormone receptor in Drosophila demonstrates a high degree of conservation of CRF-like signaling.

Endogenous biological clocks are widespread regulators of behavior and physiology, allowing for a more efficient allocation of efforts and resources over the course of a day. The extent that different processes are regulated by circadian oscillators, however, is not fully understood. We investigated the role of the circadian clock on short-term associative memory formation using a negatively reinforced olfactory-learning paradigm in Drosophila melanogaster. We found that memory formation was regulated in a circadian manner. The peak performance in short-term memory (STM) occurred during the early subjective night with a twofold performance amplitude after a single pairing of conditioned and unconditioned stimuli. This rhythm in memory is eliminated in both timeless and period mutants and is absent during constant light conditions. Circadian gating of sensory perception does not appear to underlie the rhythm in short-term memory as evidenced by the nonrhythmic shock avoidance and olfactory avoidance behaviors. Moreover, central brain oscillators appear to be responsible for the modulation as cryptochrome mutants, in which the antennal circadian oscillators are nonfunctional, demonstrate robust circadian rhythms in short-term memory. Together these data suggest that central, rather than peripheral, circadian oscillators modulate the formation of short-term associative memory and not the perception of the stimuli.

Near-infrared spectroscopy offers the potential for direct in situ analysis in complex biological systems. Chemical selectivity is a critical issue for such measurements given the extent of spectral overlap of overtone and combination spectra. In this work, the chemical basis of selectivity is investigated for a set of multivariate calibration models designed to quantify glucose, glucose-6-phosphate, and pyruvate independently in ternary mixtures. Near-infrared spectra are collected over the combination region (4,000-5,000 cm(-1)) for a set of 60 standard solutions maintained at 37 degrees C. These standard solutions are composed of randomized concentrations (0.5-30 mM) of glucose, glucose-6-phosphate, and pyruvate. Individual calibration models are constructed for each solute by using the partial least-squares (PLS) algorithm with optimized spectral range and number of latent variables. The resulting standard errors are 0.90, 0.72, and 0.32 mM for glucose, glucose-6-phosphate, and pyruvate, respectively. A pure component selectivity analysis (PCSA) demonstrates selectivity for each solute in these ternary samples. The concentration of each solute is also predicted for each sample by using a set of net analyte signal (NAS) calibration models. A comparison of the PLS and NAS calibration vectors demonstrates the chemical basis of selectivity for these multivariate methods. Selectivity of each PLS and NAS calibration model originates from the unique spectral features associated with the targeted analyte. Overall, selectivity is demonstrated for each solute with an order of sensitivity of pyruvate > glucose-6-phosphate > glucose.