Current Pharmaceutical Biotechnology - Volume 6, Issue 5, 2005

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

Spirulina, a filamentous cyanobacterium, possesses diverse biological activities and nutritional significance due to high concentration of natural nutrients, having bio-modulatory and immuno-modulatory functions. Different Spirulina preparations influence immune system viz. increase phagocytic activity of macrophages, stimulating the production of antibodies and cytokines, increase accumulation of NK cells into tissue and activation and mobilization of T and B cells. Spirulina have also shown to perform regulatory role on lipid and carbohydrate metabolism by exhibiting glucose and lipid profile correcting activity in experimental animals and in diabetic patients. Preparations have been found to be active against several enveloped viruses including herpes virus, cytomegalovirus, influenza virus and HIV. They are capable to inhibit carcinogenesis due to anti-oxidant properties that protect tissues and also reduce toxicity of liver, kidney and testes.

During the last years, Fluorescence Correlation Spectroscopy (FCS) has proven to be a powerful tool for basic research in many applications. The combination of a minimal detection volume in the femtoliter range coupled with very high sensitivity extends the possibilities to design sensitive homogeneous tests. In this article we illustrate the analysis of binding processes with FCS based on the changes in diffusion characteristics of GFP upon binding to an antibody. Problems induced by highly heterogeneous samples are discussed and differences of GFP binding to a monoclonal and a polyclonal antibody are shown and analyzed. We stress data processing, limitations and useful approximations in FCS methodology. Basic ideas of data acquisition and processing as well as new developments and applications are presented.

Diabetes is a syndrome of disordered metabolism and inappropriate hyperglycemia resulting from a deficiency of insulin secretion or insulin resistance. Insulin, a pancreatic hormone, helps to lower the blood sugar levels. The structural features of insulin and insulin receptors are summarized. Diabetic patients use insulin in the form of injections, which involves lots of pain, and a need for non-invasive, alternative mode of insulin administration is desired. These challenges have lead to attempts in insulin therapy using oral, nasal, pulmonary, rectal, transdermal, buccal, gene therapy, islet cell transplantation and diabetes vaccine. Among all the approaches pulmonary administration has achieved some clinical significance. Future approaches that can be exploited for insulin therapy in Insulin Dependent Diabetes Mellitus [IDDM] have been summarized. Insulin inhalers or tablets for IDDM are interesting alternatives.

Bioreactors play a key role in the field of biologics, where they are used for the production of recombinant therapeutic proteins by large-scale cultivation of animal cells. There are several types of bioreactors, including stirred-tank, airlift, hollow-fiber, and Rotary Cell Culture System (RCCS) designs. The stirred-tank bioreactor is one of the most commonly used types, and is used both for industrial applications and laboratory research. The RCCS, invented by NASA, is increasingly used in the area of tissue engineering for medical purposes. Important improvements have been made in the design of traditional bioreactors, and new types of bioreactor are also being developed such as Couette-Taylor bioreactor, multifunctional-membrane bioreactor, and shaking bioreactor. Work is also progressing on techniques to improve the performance of bioreactors, including perfusion culture, the use of microcarriers, and methods of suppressing apoptosis and of monitoring cell growth in real time. Given the demand for the production by animal cells for use in the growing number of clinical applications, further advances in bioreactor technology can be expected during the next few years. Two main goals will be pursued: firstly, to increase output by high density cultivation of animal cells to produce high value protein pharmaceutics or viral vectors for clinical gene therapy; and secondly, to create a three-dimension space similar to that of an in vivo environment to regenerate tissue or organ and to reproduce valuable cells that are hard to culture in the traditional culture system.

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.