Two photolabile thioglycosides (8 and 9) were synthesized by Koenigs-Knorr type glycosylation. These compounds, being enzyme-resistant analogues of N-acetylhexosaminides, were shown to be good competitive inhibitors of lysosomal beta-hexosaminidase (2-acetamido-2-deoxy- beta-D-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) action. For photoaffinity labeling 3H-labeled 8a was prepared by enzymatic oxidation with galactose oxidase followed by reduction with sodium [3H]borohydride. Compound 8a, when photolyzed in the presence of hexosaminidase, specifically labeled both subunits of the enzyme.

Affinity Labels↗

PubMedDate not supplied

First steps in robot automation of sampling and sample management during cultivation of mammalian cells in pilot scale.

The robot automation of sampling and the subsequent treatment and storage of aliquots during mammalian cell cultivations was investigated. The complete setup, the development and testing of the sampling device, the robot arm, and the cell imaging system are described. The developed sampling device is directly coupled to a pilot bioreactor. It allows the computerized sterile filling of cell broth into 50 mL sample tubes. After each sampling the whole tubing system is steam sterilized. For further off-line treatment a robot takes the sample to the different devices. This robot is equipped with a camera and a force/torque sensor. A color-based object recognition guides the arm in a complex surrounding with different illumination situations, enabling the robot to load the sampling device with tubes and take the sample to further devices. For necessary pipetting and refilling we developed a computerized device. Cells are automatically stained and counted using an imaging system. The cell number and viability is automatically saved in a process control system together with the on-line parameters. During several cultivations in 20 and 100 L scale these main components of the automation strategy were successfully tested.

Animals↗

PubMedDate not supplied

Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor.

For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH-R) in Graves' disease based on a novel coated tube assay system, human TSH-R is needed in large amounts. Whereas expression of TSH-R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH-R, but only in very low amounts. Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor. Here we report on the development of a serum-free production-scale process based on a stable transformed and highly productive human leukemia cell line K562 (1). Starting with K562-TSH-R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum-free medium. The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days. The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The process development was based on cultivations in a 2-L bench-scale bioreactor. Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device were compared. After process optimization a continuous process using spin-filter was set up and run in a 20 L-pilot-scale bioreactor. The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH-R in Graves' disease.

Bioreactors↗

PubMedDate not supplied

Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effects on recombinant human antithrombin III integrity.

Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human recombinant antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha (2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha (2-->3)Gal- and Gal beta (1-->4)-GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal recombinant protein concentration is highly recommended to avoid product desialylation.

Animals↗

PubMedDate not supplied

The hNT human neuronal cell line survives and migrates into rat retina.

The present studies were undertaken to determine if hNT cells can survive in the vitreous of the eye and migrate into the retina. The hNT neuronal cell line represents a uniform source of human tissue that may be of use in retinal grafts. hNT cells stored in liquid nitrogen were thawed and labeled with the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Thirty thousand cells in 1 microL were injected epiretinally in rat. At survival times of 3, 14, 28, or 56 days, retinal sections were examined quantitatively by epifluorescence to reveal DiI-labeled cells. hNT cells survived in the vitreous at all time points without evidence of vascularization. At 3 days, essentially no hNT cells were found in deep retina, and only very few were attached to retina. At days 14, 28, and 56, hNT cells were found to cluster on the vitreal/retinal interface, and in deeper layers. The clusters of hNT cells took on the shape of a funnel at 14 days, and inverted funnel at 28 days, and by 56 days, populated the photoreceptor layer as a stratum. It is possible that hNT cells took on the morphology and function of photoreceptors. These results suggest that hNT cells injected epiretinally survive in the vitreous at least 56 days, migrate to the retinal/vitreous interface, and may migrate through the retina. This system permits the independent and quantitative evaluation of survival and migratory trophic responses.

Animals↗

PubMedDate not supplied

Screening, diagnosis and education of women with gestational diabetes.

A study was conducted of the screening, diagnosis and education of women with gestational diabetes in South Australia. This paper provides a review of the relevant literature and a brief summary of the study, together with an overview of a patient education initiative introduced as a consequence of the study findings.

Diabetes, Gestational↗