Pam Bounelis

Glucosamine administration during resuscitation improves organ function after trauma hemorrhage.

ABSTRACT Stress-induced hyperglycemia is necessary for maximal rates of survival after severe hemorrhage; however, the responsible mechanisms are not clear. One consequence of hyperglycemia is an increase in hexosamine biosynthesis, which leads to increases in levels of O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) on nuclear and cytoplasmic proteins. This modification has been shown to lead to improved survival of isolated cells after stress. In view of this, we hypothesized that glucosamine (GlcNH2), which more selectively increases the levels of O-GlcNAc administration after shock, will have salutary effects on organ function after trauma hemorrhage (TH). Fasted male rats that underwent midline laparotomy were bled to a mean arterial blood pressure of 40 mmHg for 90 min and then resuscitated with Ringer lactate (four times the shed blood volume). Administration of 2.5 mL of 150 mmol L−1 GlcNH2 midway during resuscitation improved cardiac output 2-fold compared with controls that received 2.5 mL of 150 mmol L−1 NaCl. GlcNH2 also improved perfusion of various organs systems, including kidney and brain, and attenuated the TH-induced increase in serum levels of IL-6 (902 ± 224 vs. 585 ± 103 pg mL−1) and TNF-&agr; (540 ± 81 vs. 345 ± 110 pg mL−1) (values are mean ± SD). GlcNH2 administration resulted in significant increase in protein-associated O-GlcNAc in the heart and brain after TH. Thus, GlcNH2 administered during resuscitation improves recovery from TH, as assessed by cardiac function, organ perfusion, and levels of circulating inflammatory cytokines. This protection correlates with enhanced levels of nucleocytoplasmic protein O-GlcNAcylation and suggests that increased O-GlcNAc could be the mechanism that links stress-induced hyperglycemia to improved outcomes.

Identification of a human cDNA with high homology to yeast omnipotent suppressor 45

Omnipotent suppression is a well-established phenomenon in yeast and bacteria in which nonsense mutations are misread. Wild-type (wt) suppressors are presumed to be involved in ensuring the fidelity of translation. We report a human homolog to wt yeast omnipotent suppressor 45 which shares 63% identity at the nucleotide level in the area of open reading frame (ORF) and 73% similarity at the amino acid (aa) level. The aa sequence of the human protein was deduced from a 2.3-kb cDNA (TB3-1) isolated from an adenocarcinoma T84 cell line cDNA library. The cDNA contains an ORF of 1284 bp which encodes a 47.8-kDa protein. Two transcripts for the clone were identified (2.6 and 4.0 kb) in a variety of human cell types. The strong structural similarity to yeast omnipotent suppressor 45, and its widespread expression suggest that this cDNA may play a role in the accurate recognition of nonsense codons in mammalian cells.

The Addition of Glucose‐1‐Phosphate to the Cytoplasmic Glycoprotein Phosphoglucomutase Is Modulated by Intracellular Calcium in PC12 Cells and Rat Cortical Synaptosomes

Abstract: In a number of different cell types, phosphorylation of a 63‐kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus‐sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester‐linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [14C]glucose or the [β‐35S]phosphorothioate analogue of UDP‐glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.

Chromosomal localization of a human cDNA containing a DIDS binding domain and demonstrating high homology to yeast omnipotent suppressor 45

We recently have identified a full-length cDNA (TB3-1) from a human adenocarcinoma cell line T84 cDNA library that encodes a 47.8-kDa protein. TB3-1 shares identity with the putative yeast translation termination factor omnipotent suppressor 45. Using human-mouse somatic cell panel analysis, a family of sequences with high homology to the TB3-1 cDNA clone were localized to human chromosomes 5, 6, 7, and X. Southern analysis of a panel of mammalian and chicken genomic DNA demonstrates that TB3-1 is well conserved in higher vertebrates.

Phosphoglucomutase is a Cytoplasmic Glycoprotein Implicated in the Regulated Secretory Pathway

Phosphorylation can have a profound effect on a protein’s functional capabilities. One strategy for identifying proteins whose behaviors change in response to an external stimulus is to determine those in which changes in phosphorylation correlate with the presentation of the stimulus. In Paramecium tetraurelia, which exhibits a regulated secretory pathway, this strategy led to the isolation of parafusin (Gilligan and Satir, 1982), a 63 kDa cytoplasmic phosphoprotein that loses its phosphate upon stimulation with secretagogue. It then regains its phosphate within seconds thereafter (Ziesness and Plattner, 1985).