Biomedical subjects

D G Fast

Publications and source records attributed to D G Fast.

5 recordsLinked to original sources

PubMed2001

Rapid imaging of human melanoma xenografts using an scFv fragment of the human monoclonal antibody H11 labelled with 111In.

H11 is a human IgM monoclonal antibody which recognizes a novel tumour-associated antigen expressed on melanoma, glioma, breast cancer, colon cancer, prostate cancer, lung cancer and B-cell lymphoma. In this study, a recombinant single-chain Fv (scFv) fragment of H11 labelled with 111In was investigated for tumour imaging in athymic mice implanted subcutaneously with A-375 human melanoma xenografts. H11 scFv was derivatized with diethylenetriaminepentaacetic acid (DTPA) for labelling with 111In. The immunoreactivity of DTPA-H11 scFv against A-375 cells in vitro ranged from 23% to 36%. 111In-DTPA-H11 scFv was rapidly eliminated from the blood and most normal tissues (except the kidneys) reaching maximum tumour/blood ratios of 12:1 at 48 h post-injection. Tumours were imaged as early as 40 min after injection. The kidneys accumulated the highest concentration of radioactivity (up to 185% injected dose/g). Tumour uptake was 1-3% injected dose/g. The whole-body radiation absorbed dose predicted for administration of 185 MBq of 111In-DTPA-H11 scFv to humans was 37 mSv. The radiation absorbed dose estimates for the kidneys, spleen and intestines were 405 mSv, 698 mSv and 412 mSv, respectively. The results of this preclinical study and a concurrent phase I trial suggest a promising role for H11 scFv for tumour imaging.

Animals↗

PubMed1997

The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion.

UNLABELLED: Secretion of apolipoprotein B (apoB) from mammalian cells requires the presence of functional microsomal triglyceride transfer protein (MTP). We previously reported that co-expressing the human intestinal form of apoB, B48, with both subunits of human MTP in oleate-treated Sf21 cells led to a dramatic induction of B48 secretion. Deletion mutagenesis studies showed that the cysteine-enriched amino terminus of apoB was necessary for the MTP responsiveness (Gretch, D. G., Sturley, S. L., Wang, L., Dunning, A., Grunwald, K. A. A., Wetterau, J. R., Yao, Z., Talmud, P., and Attie, A. D. (1996) J. Biol. Chem. 271, 8682-8691). We therefore hypothesized that the small subunit of MTP, protein-disulfide isomerase (PDI), plays a role in apoB secretion by facilitating correct disulfide bond formation. To determine whether the enzymatic activities of PDI are important for MTP-stimulated apoB secretion, the wild type PDI subunit was replaced with an active site mutant, mPDI (Cys36 --> Ser/Cys380 --> Ser), lacking both disulfide shuffling and redox activities. MTP containing mPDI was fully functional in promoting apoB and triglyceride secretion. Therefore, the shufflase and redox activities of PDI are not necessary for the function of MTP. Since PDI exists in large molar excess over the other subunit of MTP, the role of free PDI (independent of the MTP complex) was investigated. PDI or mPDI was co-expressed with B48 and B17, a fragment encompassing the amino-terminal 17% of apoB. Mutant PDI significantly and specifically reduced the accumulation of the B17 and B48 both intracellularly and in the culture medium. The reduction was partially eliminated by the protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, consistent with rapid co- or post-translational degradation of apoB in the presence of mPDI. Treating the cells with oleate reversed the effect of mPDI on B48 secretion in a dose-dependent manner, but had no effect on B17. IN CONCLUSION: 1) the role of PDI in the MTP complex involves functions other than its known enzymatic activities; 2) one or both of the enzymatic activities of free PDI is/are important for the MTP-independent steps of apoB secretion; 3) oleate can affect apoB secretion at high physiological concentrations and compensate for the insufficiency of PDI activities.

Animals↗

PubMed1995

Nascent VLDL phospholipid composition is altered when phosphatidylcholine biosynthesis is inhibited: evidence for a novel mechanism that regulates VLDL secretion.

Previous work has shown that inhibition of phosphatidylcholine biosynthesis inhibits very low density lipoprotein (VLDL) secretion by causing a decrease in the number of particles in the Golgi but not in the endoplasmic reticulum of rat liver (Verkade et al. (1993) J. Biol. Chem. 268, 24,990-24,996). One explanation for this observation was that VLDL from choline deficient livers was degraded in a post-endoplasmic reticulum compartment. This hypothesis was supported by experiments in which choline deficient (CD) or choline supplemented (CS) rat hepatocytes were incubated +/- Brefeldin A. In the presence of Brefeldin A, VLDL secretion was blocked, but no difference was observed in the degradation of apolipoprotein B (apoB) within the CD or CS cells. If increased catabolism of apoB were occurring in the endoplasmic reticulum of CD hepatocytes, enhanced degradation of apoB in CD cells might have been expected. Inhibition of phosphatidylcholine biosynthesis also caused decreases in the phosphatidylcholine content of membranes of the secretory pathway. The lipids of nascent VLDLs from the lumina of endoplasmic reticulum and Golgi prepared from CD rat liver were relatively enriched in phosphatidylethanolamine and depleted of phosphatidylcholine when compared to samples from CS liver. The changes in nascent VLDL phospholipid composition mimicked that of the organelle membranes from which the VLDLs were isolated. Possibly the phospholipid composition of the organelles is a factor in determining the final phospholipid composition of VLDLs. One hypothesis is that when phosphatidylcholine biosynthesis is impaired, nascent VLDL is assembled incorrectly and degraded by a quality control protease in a post-endoplasmic reticulum compartment.

Animals↗

PubMed1993

Impaired biosynthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum of rat liver.

We have investigated the mechanism by which inhibition of phosphatidylcholine biosynthesis in rat hepatocytes by choline deprivation causes a reduction in the secretion of very low density lipoprotein (VLDL) (Yao, Z., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004). Rats ingested a choline-deficient or control diet for 3 days, and subcellular fractions of liver were prepared. No change in the amount of apolipoprotein B in the lumina of the endoplasmic reticulum was observed, but there was a 40-50% decrease of apolipoprotein B in the lumina of the Golgi from choline-deficient compared with control rats. Incubation of microsomes, derived from choline-deficient and -supplemented hepatocytes, with trypsin showed similar degradation of apolipoprotein B, indicating similar quantities of this protein are present on the surface and within the lumina. The VLDL particles in the Golgi of liver cells and in plasma, on average, were larger in samples derived from choline-deficient compared with choline-supplemented animals. Incubation of plasma VLDL with proteases demonstrated that the apolipoprotein B of plasma VLDL particles from choline-deficient animals had a different susceptibility to digestion than did VLDL from choline-supplemented animals. These data indicate that the number of VLDL particles assembled in the endoplasmic reticulum of liver is similar in choline-deficient and -supplemented rats, but the number of particles is decreased in the Golgi from choline-deficient animals.

Animals↗

PubMed1993

The role of the carbohydrate chains of Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase for enzyme activity.

Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2,6 sialyltransferase, EC 2.4.99.1) is a glycoprotein containing carbohydrate chains of the complex type (Jamieson, J.C. (1989) Life Sci. 43, 691-697). The carbohydrate chains may be important for controlling the expression of sialyltransferase catalytic activity during transit of the enzyme from the rough endoplasmic reticulum to the Golgi complex where it is active as a membrane bound enzyme anchored to the luminal face. To study the role of the carbohydrate chains of sialyltransferase for enzyme activity, conditions were established in which the native enzyme was deglycosylated with N-Glycanase and endo F. It was found that Glycanase removed the carbohydrate chains from native sialyltransferase, but methanol or ethanol had to be present for rapid and complete deglycosylation. Presence of methanol or ethanol were not essential for removal of carbohydrate chains with endo F. There was a correlation between the loss of catalytic activity of sialyltransferase with increased deglycosylation. After deglycosylation with Glycanase for 18 h catalytic activity was largely eliminated and there was a reduction in molecular mass of about 5 kDa compared to the untreated enzyme when examined by immunoblot analysis; this reduction was identical to that found when the denatured enzyme was deglycosylated with Glycanase. At shorter times of incubation partially deglycosylated forms of the enzyme were detected. Complete deglycosylation of native or denatured sialyltransferase with endo F could not be achieved. However, incubation with endo F for 24 h resulted in a loss of catalytic activity of about 60%. Immunoblot analysis showed the presence of three forms of the enzyme corresponding in molecular mass to the native and deglycosylated enzyme and a third form corresponding to a partially deglycosylated enzyme. Sialyltransferase was also subjected to sequential treatment with exoglycosidases. Removal of NeuAc and Gal had little effect on catalytic activity, but subsequent removal of GlcNAc resulted in a significant loss in catalytic activity suggesting that the presence of the trimannose core with GlcNAc attached is important for the expression of catalytic activity. The presence of organic solvents during deglycosylation with Glycanase may be a useful method that can be applied to other glycoproteins.

Carbohydrate Sequence↗