Current
Pharmaceutical Biotechnology
ISSN: 1389-2010
Current Pharmaceutical Biotechnology
Volume 6, Number 5, October 2005
Contents
Comparison of Different Fluorescence Fluctuation Methods for
their Use in FRET Assays: Monitoring a Protease Reaction Pp.351
C. Eggeling, S. Jäger, D. Winkler and Peet Kask
[Abstract] [Purchase
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Nutritional and Therapeutic Potential of Spirulina
Pp.373
Z. Khan, P. Bhadouria and P.S. Bisen
[Abstract] [Purchase
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Diffusion and Binding Properties Investigated
by Fluorescence Correla-tion Spectroscopy (FCS) Pp.381
D. Grünwald, M.C. Cardoso, H. Leonhardt and V. Buschmann
[Abstract] [Purchase
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Advanced Approaches in Insulin Delivery Pp.387
I.M. Shaikh, K.R. Jadhav, S. Ganga, V.J. Kadam and S.S.
Pisal
[Abstract] [Purchase
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The Bioreactor: A Powerful Tool for Large-Scale
Culture of Animal Cells Pp.397
D. Wang, W. Liu, B. Han and R. Xu
[Abstract] [Purchase
Issue/Articles]
Enhancing the Sensitivity of Fluorescence Correlation
Spectroscopy by Using Time-Correlated Single Photon Counting
Pp.405
D.C. Lamb, B.K. Müller and C. Bräuchle
[Abstract] [Purchase
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Abstracts
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Comparison of Different Fluorescence Fluctuation
Methods for their Use in FRET Assays: Monitoring a Protease
Reaction
C. Eggeling, S. Jäger, D. Winkler and
Peet Kask
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 sig-nificantly 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 normal-ized
correlation data when combining any fluorescence correlation
method with FRET, since the analysis of the unnor-malized
data introduces more accuracy and is less sensitive to the
experimental drawbacks.
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Nutritional and Therapeutic Potential of Spirulina
Z. Khan, P. Bhadouria and P.S. Bisen
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.
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Diffusion and Binding Properties Investigated by Fluorescence
Correla-tion Spectroscopy (FCS)
D. Grünwald, M.C. Cardoso, H. Leonhardt and V. Buschmann
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.
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Advanced Approaches in Insulin Delivery
I.M. Shaikh, K.R. Jadhav, S. Ganga, V.J. Kadam and S.S.
Pisal
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.
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The Bioreactor: A Powerful Tool for Large-Scale Culture
of Animal Cells
D. Wang, W. Liu, B. Han and R. Xu
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
sup-pressing 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.
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Enhancing the Sensitivity of Fluorescence Correlation
Spectroscopy by Using Time-Correlated Single Photon Counting
D.C. Lamb, B.K. Müller and C. Bräuchle
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
fluo-rescence resonance energy transfer (FRET), quantitative
FCCS measurements can be performed in spite of the FRET and
the static FRET efficiency can be determined.
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