A mechanical model for the human intervertebral disc

Stress relaxation experiments were performed on specimens from a human intervertebral disc. Specimens were made from the nucleus pulposus and from the external lamellae of the anulus fibrosus in two different orientations. Tests were run with varying moisture content so as to develop a relaxation master curve. A model was developed based on the experimental data. It was found that the short term master curve for the lamellae of the anulus and nucleus are similar, whereas the long term rubbery plateau is different between the lamellae and the nucleus. It was also established that the master curves for different lamellae and the nucleus were shifted relative to each other in the time domain due to changes in water content. The average relaxation modulus of the whole disc was obtained by averaging the properties between the anulus and nucleus. This model was then used for studies of Schmorl's nodes, of degenerated discs and for circumstances in which hydration is considered to be important.     

Genomic organization and expression of hamster UDP-N-acetylglucosarmine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase

The hamster gene for uridine diphosphate N-acetyl-D-glucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase(L-G1PT) was found to extend over 6.5 kb and to contain nineexons. The exons ranged in size from 63 to 214 bp, encodingthe 408 amino acid protein. The introns ranged from 85 bp to1.4 kb. Upstream 5' sequences included two possible TATA boxes,one possible CCAAT box and at least two potential GC boxes.Heterologous expression was successful in Schizosaccharomycespombe, and resulted in cells that were tunicamycin resistantand had 12-fold more L-G1PT activity than wild-type cells. Antiserumprepared to a hydrophilic peptide (residues 300–320) ofthe L-G1PT protein reacted with a 35–36 kDa protein inmembrane samples from Chinese hamster ovary (CHO) cells andS.pombe cells that had increased levels of L-G1PT activity.In both cases, antigenic peptide competed with the 35–36kDa protein detected by the antiserum. N-acetylglucosarmine 1-phosphate transferase dolichol glycosylation     

Aspartic acid 252 and asparagine 185 are essential for activity of lipid N-acetylglucosaminylphosphate transferase

A key step in the assembly of oligosaccharide-lipid intermediatesin N-linked glycosylation is the transfer of N-acetylglucosamine1-phosphate to dolichyl phosphate, catalyzed by the enzyme UDP-N-acetylglucosaminyl:dolichylphosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT).Comparison of the amino acid sequences of L-G1PT from five diversespecies showed 75 amino acids identical in all five proteins.Using site-directed mutagen-esis, we analyzed the importanceof a number of these conserved residues to the enzymatic activityof L-G1PT using a plasmid shuffling procedure in Schizosaccharomycespombe. S.pombe cells containing a chromosomal deletion of theessential gpt⁺ gene are rescued by a plasmid containing theS.pombe gpt open reading frame. Replacement of that plasmidby a plasmid encoding a mutated hamster L-G1PT cDNA sequenceindicated that the mutated protein provided sufficient enzymeactivity to permit cell growth. Mutations of aspartic acid 252and asparagine 185 did not allow plasmid shuffling, indicatingthese residues were essential for activity. A combination ofmutations at asparagine 182 and tryptophan 122 did not allowplasmid shuffling, although the single mutations did. Overexpressionof the mutant proteins in S.pombe conferred tunicamycin (TM)resistance, indicating that the mutant proteins had a conformationnecessary for binding TM, a substrate analog. The mutant proteinswere also detected in Western blots and were correctly localizedto the membrane fractions. However, the overexpressed proteinsdid not increase the endogenous level of enzymatic activityin these cells, indicating they were enzymatically inactive. N-acetylglucosaminylphosphate transferase mutagenesis plasmid shuffling Schizosaccharomyces pombe tunicamycin