Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells.

Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells.

The neuroprotective and neurorescue effects of carbamylated erythropoietin Fc fusion protein (CEPO-Fc) in a rat model of Parkinson's disease.

Parkinson's disease is characterized by progressive degeneration of dopaminergic neurons. Thus the development of therapeutic neuroprotection and neurorescue strategies to mitigate disease progression is important. In this study we evaluated the neuroprotective/rescue effects of erythropoietin Fc fusion protein (EPO-Fc) and carbamylated erythropoietin Fc fusion protein (CEPO-Fc) in a rat model of Parkinson's disease.

Recombinant IgA production: single step affinity purification using camelid ligands and product characterization.

Immunoglobulins of isotype A (IgA) mediate a key role in mucosal immunity and are promising new immunotherapeutic candidates, but difficulties in obtaining enough material often hamper their in vivo exploration. We established recombinant Chinese hamster ovary (CHO) cells which stably expressed two IgA1 antibodies under serum-free conditions. The two cell lines achieved significantly different specific productivities of 16 pg per cell and day and 100 times less, a common phenomenon in recombinant antibody expression which challenges the production and purification process.

Partial humanization and characterization of an anti-idiotypic antibody against monoclonal antibody 2F5, a potential HIV vaccine?

We recently developed a murine anti-idiotypic antibody (Ab2/3H6) versus the human monoclonal antibody 2F5, one of a few antibodies yet known to neutralize a broad range of HIV-1 primary isolates. Ab2/3H6 was not only able to bind to the paratope of mAb 2F5 but also significantly inhibited the binding of 2F5 to its synthetic epitope ELDKWA on gp41. In the present work we describe the partial humanization, expression, and characterization of Ab2/3H6 variants followed by several corresponding interaction studies with 2F5.

Applicability of different fluorescent dyes for isoform quantification on linear IPG gels.

For biotechnological research, development, and production various analytical methods are required to determine the quality of the target product. In this context, the determination of isoforms is state-of-the-art; however, the majority of applied techniques are more qualitative than quantitative. To address this fact, we evaluated different post- and pre-electrophoretic staining dyes for their applicability on linear IPG gels using recombinant human erythropoietin as a model protein.

Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells.

One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG(1) molecule.

A novel strategy for quantitative isoform detection directly performed from culture supernatant.

Currently, one of the most used techniques for the determination of isoform pattern analysis is isoelectric focusing. Routinely, this is performed by immunoblotting. Blotting of proteins after isoelectric focusing on IPG gels may cause several problems, such as protein loss by the blotting itself and band broadening, in some cases the immunostaining with antibodies might be problematic.

Process parameter shifting: Part II. Biphasic cultivation-A tool for enhancing the volumetric productivity of batch processes using Epo-Fc expressing CHO cells.

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields.

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors.

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line.

Characterization of molecular features, antigen-binding, and in vitro properties of IgG and IgM variants of 4E10, an anti-HIV type 1 neutralizing monoclonal antibody.

The human monoclonal antibody 4E10 has been generated previously by immortalization of peripheral blood cells from an HIV-1-infected individual. This antibody binds to the linear epitope NWFDIT on gp41 and exhibits exceptional neutralizing activity against a broad spectrum of primary HIV-1 isolates. In the present study, molecular features, immunoreactivity, and functional activity of 4E10 were studied.

Anti-idiotypic antibody Ab2/3H6 mimics the epitope of the neutralizing anti-HIV-1 monoclonal antibody 2F5.

Anti-idiotypic antibodies directed against potentially neutralizing anti-HIV-1 antibodies may mimic epitopes on gp41 otherwise cryptic to the immune system. This study reports the generation of murine monoclonal antibody Ab2/3H6 blocking the binding of human Ab1 2F5 to the synthetic epitope and to gp160 in an enzyme-linked immunosorbent competition assay. Ab2/2H6 diminished the neutralizing potency of 2F5 in an in-vitro neutralization assay.

[Ocular manifestation of progressive essential telangiectases].

Generalized essential telangiectasia is a rare skin disorder characterized by the development of dilated venules beginning at the lower extremities and progressively spreading out to the rest of the body. It is not related to any known affection and is therefore considered to be essential. The condition tends to affect mostly women in their midthirties.

Rational design of an improved induction scheme for recombinant Escherichia coli.

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters.

Recombinant expression of alliin lyase from garlic (Allium sativum) in bacteria and yeasts.

Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors. The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm.

[Neurophysiologic follow-up in patients with hypophyseal adenoma operations].

Background: Pituitary tumors are adenomas of a region of the sella turcica which can produce compression of the anterior visual pathway.

Patients And Methods: Besides clinical signs of visual improvement such as enhanced visual acuity and visual field, the use of electroophthalmological methods can help monitor patients after neurosurgery.

Results: The N75 P100 amplitude of the p-VEP has proven to be the most sensitive marker for measuring-improvements after surgery.

[Is cranial corneoscleral 6 mm "no-stitch" tunnel incision contraindicated in against-the-rule astigmatism?].

Background: There is a risk to increase a preexisting astigmatism against the rule by performing the incision at the 12 o'clock position.

Patients And Methods: In the prospective study 15 patients with a preoperative astigmatism over 0.25 D axis 90 degrees +/- 15 degrees the first cataract extraction was performed by a cranial, the operation of the other eye by a temporal 6-mm no-stitch tunnel incision.

Optimization of recombinant gene expression in Escherichia coli.

The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor.

[Value of electrophysiology in ophthalmologic monitoring of patients with endocrine orbitopathy].

Background: Patients with Graves' ophthalmopathy may suffer from optic nerve involvement. Different ophthalmological examinations reveal the stage of this disease.

Patients And Methods: In a retrospective study 27 patients with Graves' ophthalmopathy were examined.

[Dapiprazol antagonizes tropicamide- and phenylephrine-induced mydriasis in the elderly].

Dapiprazole eye drops were applied 1 h after topical tropicamide or phenylephrine instillation in 30 elderly subjects. Pupil diameter normalized much earlier than in controls receiving placebo. Dapiprazole constricted the tropicamide-dilated pupil by 1 mm after 1 h and normalized the pupil diameter of the phenylephrine-dilated pupil within 1 h, whereas the pupils of the patients receiving placebo remained dilated.

Internal Ca2+ ions inactivate and modify ATP-sensitive potassium channels in adult mouse skeletal muscle.

The effects of internal Ca2+ ions on single ATP-sensitive potassium channels (KATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel activity was high when patches were excised in a Ca(2+)-free, Na(+)-rich solution and declined irreversibly within seconds in the presence of internal Ca2+ (0.1 and 2 mM).

Isoproterenol enhances a calcium-independent potassium current in mouse anterior pituitary tumor cells.

1. The patch-clamp technique was used to study the action of the beta-adrenergic agonist (-)-isoproterenol in anterior pituitary tumor cells of the mouse. 2.

Vanadate as an activator of ATP--sensitive potassium channels in mouse skeletal muscle.

The inside-out mode of the patch-clamp technique was used to study adenosine-5'-triphosphate (ATP)-sensitive K+ channels in mammalian skeletal muscle. Vanadate, applied to the cytoplasmic face of excised patches, was a potent activator of ATP-sensitive K+ channels. Divalent cations (Mg2+, Ca2+) were a prerequisite for the activating process.