In vitro detection of adrenocorticotropic hormone levels by fluorescence correlation spectroscopy immunoassay for mathematical modeling of glucocorticoid-mediated feedback mechanisms

Performing quantitative, highly sensitive measurements at a single molecule level is often necessary to address specific issues related to complex molecular and biochemical systems. For that purpose, we present a technique exploiting both the flexibility of immunoassays as well as the low operating costs and high throughput rates of the fluorescence correlation spectroscopy (FCS) method. That way we have established a quantitative measurement technique providing accurate and flexibly time resolved data of single molecules. Nanomolar changes in adrenocorticotropic hormone (ACTH) levels have been detected in a short time-frame that are caused by fast feedback actions in AtT-20 anterior pituitary glands in vitro. Especially with respect to clinical diagnostic or mathematical modeling this improved FCS setup may be of high relevance in order to accurately quantify the amounts of peptide hormones—such as ACTH—as well as signaling molecules, transcription factors, etc., being involved in intra- and extracellular reaction networks

Emerging applications of fluorescence spectroscopy in medical microbiology field

There are many diagnostic techniques and methods available for diagnosis of medically important microorganisms like bacteria, viruses, fungi and parasites. But, almost all these techniques and methods have some limitations or inconvenience. Most of these techniques are laborious, time consuming and with chances of false positive or false negative results. It warrants the need of a diagnostic technique which can overcome these limitations and problems. At present, there is emerging trend to use Fluorescence spectroscopy as a diagnostic as well as research tool in many fields of medical sciences. Here, we will critically discuss research studies which propose that Fluorescence spectroscopy may be an excellent diagnostic as well as excellent research tool in medical microbiology field with high sensitivity and specificity

Subtype-specific differences in corticotropin-releasing factor receptor complexes detected by fluorescence spectroscopy

G protein-coupled receptors have been proposed to exist in signalosomes subject to agonistdriven shifts in the assembly-disassembly equilibrium, affected by stabilizing membranelipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotrophin-releasing-factor receptors, CRFR1&2, which differ within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar to concentrationresponse curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both, CRFR1 and CRFR2, formed constitutive oligomers at the cell surface and recruited â- arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer - FRET - microscopy in living cells). However, CRFR2 - but not CRFR1 - failed to undergo agonist-induced internalization. Similarly agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy - FCS) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis (FIDA) demonstrated that the size of CRFR-complexes was not changed. Disruption of the actin cytoskeleton abolished the agonist-dependent increase in CRFR2 mobility, shifted the agonist concentration curve for CRFR2 to the left and promoted agonist-induced internalization of CRFR2. Our observations are incompatible with an agonist-induced change in monomeroligomer equilibrium, but they suggest an agonist-induced redistribution of CRFR2 into a membrane microdomain that affords rapid diffusion but restricted mobility and that is stabilized by the actin cytoskeleton. Our data show that membrane anisotropy can determine the shape and duration of receptor-generated signals in a subtype-specific manner