Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose

Through process transfer and optimization for increased antibody production to 3 g/L for a GS-CHO cell line, an undesirable drop in antibody Fc galactosylation was observed. Uridine (U), manganese chloride (M), and galactose (G), constituents involved in the intracellular galactosylation process, were evaluated in 2-L bioreactors for their potential to specifically increase antibody galactosylation. These components were placed in the feed medium at proportionally increasing concentrations from 0 to 20 × UMG, where a 1× concentration of U was 1 mM, a 1× concentration of M was 0.002 mM, and a 1× concentration of G was 5 mM. Antibody galactosylation increased rapidly from 3% at 0× UMG up to 21% at 8× UMG and then more slowly to 23% at 20× UMG. The increase was primarily due to a shift from G0F to G1F, with minimal impact on other glycoforms or product quality attributes. Cell culture performance was largely not impacted by addition of up to 20× UMG except for suppression of glucose consumption and lactate production at 16 and 20× UMG and a slight drop in antibody concentration at 20× UMG. Higher accumulation of free galactose in the medium was observed at 8× UMG and above, coincident with achieving the plateau of maximal galactosylation. A concentration of 4× UMG resulted in achieving the target of 18% galactosylation at 2-L scale, a result that was reproduced in a 1,000-L run. Follow-up studies to evaluate the addition of each component individually up to 12× concentration revealed that the effect was synergistic; the combination of all three components gave a higher level of galactosylation than addition of the each effect independently. The approach was found generally useful since a second cell line responded similarly, with an increase in galactosylation from 5% to 29% from 0 to 8× UMG and no further increase or impact on culture performance up to 12× UMG. These results demonstrate a useful approach to provide exact and specific control of antibody galactosylation through manipulation of the concentrations of uridine, manganese chloride, and galactose in the cell culture medium.     

The presequence of Arabidopsis serine hydroxymethyltransferase SHM2 selectively prevents import into mesophyll mitochondria

Serine hydroxymethyltransferases (SHMs) are important enzymes of cellular one-carbon metabolism and are essential for the photorespiratory glycine-into-serine conversion in leaf mesophyll mitochondria. In Arabidopsis (Arabidopsis thaliana), SHM1 has been identified as the photorespiratory isozyme, but little is known about the very similar SHM2. Although the mitochondrial location of SHM2 can be predicted, some data suggest that this particular isozyme could be inactive or not targeted into mitochondria. We report that SHM2 is a functional mitochondrial SHM. In leaves, the presequence of SHM2 selectively hinders targeting of the enzyme into mesophyll mitochondria. For this reason, the enzyme is confined to the vascular tissue of wild-type Arabidopsis, likely the protoxylem and/or adjacent cells, where it occurs together with SHM1. The resulting exclusion of SHM2 from the photorespiratory environment of mesophyll mitochondria explains why this enzyme cannot substitute for SHM1 in photorespiratory metabolism. Unlike the individual shm1 and shm2 null mutants, which require CO(2)-enriched air to inhibit photorespiration (shm1) or do not show any visible impairment (shm2), double-null mutants cannot survive in CO(2)-enriched air. It seems that SHM1 and SHM2 operate in a redundant manner in one-carbon metabolism of nonphotorespiring cells with a high demand of one-carbon units; for example, during lignification of vascular cells. We hypothesize that yet unknown kinetic properties of SHM2 might render this enzyme unsuitable for the high-folate conditions of photorespiring mesophyll mitochondria.     

Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity

FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice infected with the rodent malaria parasite Plasmodium vinckei.     

Establishment of a Novel In Vitro Test Setup for Electric and Magnetic Stimulation of Human Osteoblasts

When large defects occur, bone regeneration can be supported by bone grafting and biophysical stimuli like electric and magnetic stimulation (EMS). Clinically established EMS modes are external coils and surgical implants like an electroinductive screw system, which combines a magnetic and electric field, e.g., for the treatment of avascular bone necrosis or pseudarthrosis. For optimization of this implant system, an in vitro test setup was designed to investigate effects of EMS on human osteoblasts on different 3D scaffolds (based on calcium phosphate and collagen). Prior to the cell experiments, numerical simulations of the setup, as well as experimental validation, via measurements of the electric parameters induced by EMS were conducted. Human osteoblasts (3 × 10⁵ cells) were seeded onto the scaffolds and cultivated. After 24 h, screw implants (Stryker ASNIS III s-series) were centered in the scaffolds, and EMS was applied (3 × 45 min per day at 20 Hz) for 3 days. Cell viability and collagen type 1 (Col1) synthesis were determined subsequently. Numerical simulation and validation showed an adequate distribution of the electric field within the scaffolds. Experimental measurements of the electric potential revealed only minimal deviation from the simulation. Cell response to stimulation varied with scaffold material and mode of stimulation. EMS-stimulated cells exhibited a significant decrease of metabolic activity in particular on collagen scaffolds. In contrast, the Col1/metabolic activity ratio was significantly increased on collagen and non-sintered calcium phosphate scaffolds after 3 days. Exclusive magnetic stimulation showed similar but nonsignificant tendencies in metabolic activity and Col1 synthesis. The cell tests demonstrate that the new test setup is a valuable tool for in vitro testing and parameter optimization of the clinically used electroinductive screw system. It combines magnetic and electric stimulation, allowing in vitro investigations of its influence on human osteoblasts.     

Intracellular recording from a spider vibration receptor

The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ’s cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.     

Interaction of Eukaryotic Initiation Factor eIF4B with the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus Is Independent of the Polypyrimidine Tract-Binding Protein

Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.