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IN VITRO AND IN VIVO DETERMINATION OF DOPAMINE AND OTHER NEUROTRANSMITTERS USING CARBON FIBRE AND GLASSY CARBON ELECTRODES BY DIFFERENTIAL PULSE VOLTAMMETRY
No full textElectroanalysis of neurotransmitters using carbon fibre
electrodes (CFE) is very important for the development of new
drugs, for modern brain research and understanding of the molecular
processes in the central nervous system. Essential fundamental
works have been carried out by Adams [1], Gonon [2],
Pons and Fleischmann [3] concerning pretreatment of the electrodes
and techniques of measurement. However, there are still
some questions, for instance the reproducibility of electrochemical
and chemical pretreatment, influence of the kind of the
fibre, of the application of polymer layers and of other modifications.
Today commercial microelectrodes are still not avalaible.
Users of electrodes usually have developed a method to
be well applicable for themselves
MICROSCOPIC MULTICHANNEL SPECTROMETER FOR LIGHT ABSORPTION MEASUREMENTS OF PIGMENTS INSIDE OF MAMMALIAN CELLS
No full textDuring the evolution process most cells have learned to use oxygen for
establishing higher functional regulations. For this purpose different
proteins have been developed which are able to react with oxygen; as
for instance hemoglobin and myoglobin for oxygen transport and storage
or cytochromes, which act in the respiratory chain as electron carriers
to reduce oxygen to water for generating energy. Also other proteins
are known, like oxidases, which are involved in the reduction of
oxygen. The question was therefore addressed, whether monitoring of
the activity of these proteins in dependence on the oxygen partial
pressure can be used to construct a biosensor for oxygen
The Continuous Measurement of Metabolic Parameters with Electrochemical Sensors
No full textThe continuous measurement of metabolic changesis an important part of the developmentof electrochemical
sensors. Stability and sensitivity are essential requests, but compatibility towards biological
materials and interferences with other substances need consideration, too. Dueto the high demands on
the materials usable for implantation, the application to men is considerably restricted to analysis in
vitro or ex vivo
ON-LINE MONITORING OF FAST BIOPROCESSES WITH FLOW INJECTION ANALYSIS AND GASCHROMATOGRAPHY
No full textFor fundamental metabolic studies and effective control of bioprocesses on-line analysis
of substrates, products and intermediates is required. For the investigation of the metabolism
of B. subtilis on-line flow injection analysis for glucose has been applied together with a online
capillary gaschromatograph for direct analysis of meso- and D/L 2.3-butanediol, acetoin
and acetic acid in liquid samples in intervals of less than eight minutes
IDENTIFICATION OF THE RECEPTOR FOR CONTACTINHIBIN
No full textThe results in our research about contact-dependent regulation
of growth of human cells let us come to the conclusion that a
defined membrane glycoprotein we called Contactinhibin is of major
importance for a density-dependent down regulation of proliferation
in various cell cultures. Our investigations further
postulate a specific cell membrane protein receptor for
contactihibin which is defective or absent in transformed cells.
We are now trying to approach to the molecular nature and
biochemical parameters of this receptor in order to reveal its
defects in transformed cells and to find further explanations in
the phenomenon of uncontrolled tumor growth
A STRATEGY FOR ISOLATION AND STRUCTURAL ANALYSIS OF GLYCOPROTEIN OLIGOSACCHARIDES
No full textA general strategy is described for release, separation and structural analysis of Nand
O-linked glycans from glycoproteins. Following sugar analysis, to determine the
monosaccharide composition and the total carbohydrate content, the glycoprotein is
degraded by chemical and/or enzymatic methods. The N- and O-linked glycans are
separated and further fractionated into pure compoundsby gelfiltration, affinity
chromatography and high performance ion exchange chromatography. Structural
analysis is carried out by chemical analyses, fast atom bombardment mass spectrometry
and 500 MHz 1H-NMR spectroscopy. Complete structural analysis is obtained of each
glycan as well as determination of the glycosylationsites
THE EXPRESSION OF ONCOFETAL ANTIGENS ON MUCINS OF HUMAN AMNIOTIC FLUID
No full textThe relative high abundance of O-linked oligosaccharides on the
mucin-type glycoproteins has opened a favorable perspective for
detailed studies of carbohydrate epitope structures, which may be
involved in processes relevant for cellular differentiation in
development and oncogenesis. Immunological experiments, in particular
those with monoclonal antibodies raised against the cell surfaces,
have been sucessfully performed in order to monitor qualitatively
oncofetal carbohydrate antigens and the changes in their patterns
during such processes (1). On the other hand the biosynthetic studies
on glycosyltransferases responsible for assembly of complex
carbohydrates bound to proteins and their substrates lead to the
conclusion, that several enzymes compete for a common substrate of
specific structure available in the respective cellular compartment
at the certain phase of biosynthesis (2). Therefore some carbohydrate
microheterogeneity arising from the presence of different terminal
epitope structures as well as the different branch length and
different branching patterns on the single glycan attachment site
should be expected. The structures of the O-linked glycans, liberated
from the mucin-type glycoproteins by reductive elimination can be
determined by spectroscopic methods, using different techniques of
nuclear magnetic resonance (NMR) (3) and mass spectrometry (4). By
combining the immunological and spectroscopic approach numerous
oncofetal carbohydrate antigen structures have been found not only on mucins of developing systems (5), but on mucins of
human body fluids of normal individuals like in seminal plasma (6)
and milk (7) as well. Oncofetal antigens, recognized by a number of
monoclonal antibodies like C-50, NS 19-9, OC 125, Leu Ml, 49 H 8 and
115 C 2, are strongly expressed also in the mucine fraction of human
amniotic fluid (8). These were analysed by fast atom bombardment mass
spectrometry (FAB-MS) (9), in particular Suitable for the
determination of carbohydrate sequences, their branching patterns and
their molecular size in native and derivatized samples, even in
complex mixtures
OLIGOSACCHARIDE STRUCTURES OF GLYCOPROTEINS FROM RECOMBINANT MAMMALIAN CELL LINES
No full textOur group has elucidated the carbohydrate structures of a number of pharmaceutically
relevant recombinant glycoproteins (IFN-8, IL-2, t-PA, AT II, EPO as well
as mutant proteins thereof) expressed in several mammalian host cell lines (CHO,
BHK, C127, Ltk) . After enzymatic liberation of the N-linked oligosaccharide chains
by action of polypeptide:N-glycanase from the intact glycoprotein or tryptic
fragments thereof, the individual oligosaccharides were separated by a combination of
ion exchange chromatography and HPLC on NH,-phase. Oligosaccharide structures were
elucidated using several analytical techniques: GC/MS (SIM-mode) for compositional
and methylation analyses, FAB-MS for the determination of their molecular weight and
the terminal substitution pattern as well as 600 MHz 7H-NMR spectrometry for the
determination of anomeric configuration and linkage pattern of the monosaccharide
building blocks.
The recently introduced high-pH-anion-exchange-chromatography with pulsed
amperometric detection (HPAE-PAD) was applied for comparison of differently charged
oligosaccharide fractions after enzymatic desialylation, determination of oligosaccharide
structures at individual glycosylation sites and for control of batch-to-batch
consistency of biotechnologically produced glycoproteins
LIPASES : BIOTRANSFORMATIONS, ACTIVE SITE MODELS AND KINETICS
No full textLipases can be used to obtain various (chiral) intermediates. To
select a suitable hydrolytic enzyme from the increasing number of
commercially available lipases application of active-site models may
be very useful. Since the hydrolysis takes place at the interface, the
kinetics of lipase catalyzed reactions are strongly dependend upon the
quantity and quality of the interface. A newly developed dynamic
method, based on measuring the droplet-size distribution by light
scattering (Fraunhofer diffraction), has proven to be very useful to
measure the total interfacial area of a non-stabilized emulsion. In an
alternative approach lipase kinetics could be determined by using a
hollow fiber membrane reactor. Both approaches indicate that there is
a linear relationship between the rate of lipolysis and the
interfacial area. The effect of the quality of the interface on the
enzymic hydrolysis reaction is currently being studied to optimize
both the rate as well as the (stereo)selectivity of the hydrolysis
CHARACTERIZATION AND OVER-EXPRESSION OF A CLONED PSEUDOMONAS LIPASE GENE
No full textTriacylglycerollipases (E.C.3.1.1.3) are ubiquitous amongst microbes, plants, and
animals and catalyse the hydrolysis of ester bonds of triglycerides to form glycerol and
fatty acids. On the other hand, lipases catalyse the reverse esterification reaction in low
water conditions, forming glycerides from glycerol and free fatty acids. Moreover, some
lipases catalyse transesterification through the exchange ofesterified fatty acids with free
fatty acids. These reactions can either be selective toward a specific ester bond in
triglycerides or completely non-specific. Besides positional specificity, some lipases
demonstrate selectivity toward a particular fatty acid substrate.
Since lipases have wide versatility, considerable interest in the industrial uses of
lipases has recently developed. Industrial applications of lipases include enzymatic fat
splitting, accelerated cheese ripening, production of cocoa butter substitutes, and as a
detergent additive. Also, the enanatioselectivity of certain lipases offers an attractive
opportunity for the preparation of chiral intermediates for pharmaceutical syntheses.
Thelipase from Pseudomonassp. ATCC 21808is of particular interest for potential
industrial applications becauseofits high temperature optimum for enzymatic activity (65
deg. C), its thermostability (no activity loss after one week at 50 deg C in a phosphate
buffer), and activity over a broad pH range (4-9)