Deformation of the equine pelvis in response to in vitro 3D sacroiliac joint loading.

REASONS FOR PERFORMING STUDY: Sacroiliac joint injuries can cause poor performance; however, the interaction between pelvic mechanics and the sacroiliac joint is poorly understood. OBJECTIVE: To measure pelvic displacement during 3D sacroiliac joint loading. METHODS: Nine reflective triads were attached rigidly to bony prominences in sacropelvic specimens harvested from 14 horses for stereophotogrammetric analysis of triad displacements and joint kinematics. The sacrum was coupled to a load cell and mounted vertically within a material testing system (MTS). A pneumatic actuator was used to apply 90 Nm moments to the ischial arch to simulate nutation-counternutation and left and right lateral bending of the sacroiliac joints. Axial rotation of the sacrum was induced by torsion of the upper MTS fixture. Vectors of marker displacement within orthogonal planes of motion were measured during loading of the sacropelvic specimens. Comparisons in the magnitude and direction of triad displacements were made between paired left-right markers and paired loading conditions. RESULTS: Nutation-counternutation of the sacroiliac joint caused vertical displacement of the ischial tuberosities and cranial-caudal displacement of the wings of the ilium. Lateral bending induced rotational displacement within the horizontal plane of all pelvic landmarks, relative to the sacrum. Axial rotation of the sacrum caused elevation of the wing of the ilium ipsilateral to the direction of sacral rotation and depression of the contralateral ilial wing. Significant paired left-right differences occurred during most sacroiliac joint loading conditions. Comparable magnitudes of pelvic displacement were measured during nutation-counternutation, left and right lateral bending, and left and right axial rotation. CONCLUSIONS: The equine pelvis is not a rigid structure and asymmetric pelvic deformation occurs during most sacroiliac joint movements. CLINICAL RELEVANCE: Bony pelvic deformation should be considered a normal response to any sacroiliac joint movement.

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.

Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells.

Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N-glycan processing in GS-NS0 and GS-CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide-sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine-specific glycan motifs on NS0-derived recombinant proteins, such as Galalpha1,3Galbeta1,4GlcNAc. Although both CHO and NS0-derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N-glycan antennae associated with NS0-derived TIMP-1 terminated in alpha1,3-linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N-glycolylneuraminic (sialic) acid residues as compared with the N-acetylneuraminic acid variant. Inclusion of the nucleotide-sugar precursors, glucosamine (10 mM, plus 2 mM uridine) and N-acetylmannosamine (20 mM), in culture media were shown to significantly increase the intracellular pools of UDP-N-acetylhexosamine and CMP-sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP-N-acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N-glycans associated with TIMP-1 produced in CHO cells but not N-glycans associated with TIMP-1 from NS0 cells. In addition, elevated UDP-N-acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP-sialic acid content induced by N-acetylmannosamine had no effect on the overall level of sialylation of TIMP-1 produced by both CHO and NS0 cells, although the ratio of N-glycolylneuraminic acid:N-acetylneuraminic acid associated with NS0-derived TIMP-1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide-sugar metabolism can promote changes in N-glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells.

Spatially specific deficits in processing graphemic representations in reading and writing.

We report the performance of two brain-damaged subjects, HB and ML, whose spelling performance is characterized by selective impairment in processing the side of words contralateral to their brain damage. A striking feature of these patients' performance was the fact that their spelling errors in all tasks--written naming, written and oral spelling, and delayed copy transcription--almost exclusively concerned the right half of words (in the case of HB) or the left half of words (in the case of ML), regardless of length of the target response. These patterns of performance are interpreted as indicating damage at the level of the grapheme description computed in all spelling tasks. We also discuss the additional observations that HB tended to complete words with nonrandom letter sequences in misspelling the final half of the word and that ML tended to preserve the initial letter of the word (in forward but not backward spelling) even when she made errors on other letters in the initial half of the word. Finally, the relationship between these spatially specific impairments of reading and writing and their relationship to spatially specific deficits in non-lexical tasks is reviewed.