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L J Scott

Publications and source records attributed to L J Scott.

48 records · Page 3Linked to original sources
PubMed1992

Dynamics of four rat liver plasma membrane proteins and polymeric IgA receptor. Rates of synthesis and selective loss into the bile.

We have determined the half-lives and amounts per hepatocyte of the polymeric IgA receptor (pIgA-R) and four rat hepatocyte plasma membrane proteins and subsequently have predicted their rates of synthesis and possible routes of degradation. Using in vivo pulse-chase metabolic labeling with L-[35S]cysteine, we found that the pIgA-R had an apparent half-life of 1.1 h. Additional metabolic labeling experiments showed that CE9, HA4, and HA321 had apparent half-lives of 4-5 days, and dipeptidyl peptidase IV had an apparent half-life of 9 days. To quantify the amount of each protein per hepatocyte, homogenates and a standard curve of purified protein were compared by immunoblotting. We found that these proteins were present at 1-8 x 10(6) molecules/hepatocyte. The calculated rate of synthesis for pIgA-R was 1.6 x 10(6) molecules/hepatocyte/h, whereas the others were synthesized at much lower rates (0.9-5 x 10(4) molecules/hepatocyte/h). Using immunoblot analysis, we found that pIgA-R was released into bile at a rate of 30%/h (700%/day), whereas dipeptidyl peptidase IV and HA4 were released at a rate of 2-3%/day. While the majority of the loss of pIgA-R from hepatocytes occurred by release into the bile, less than 30% of the degradation of dipeptidyl peptidase IV and HA4 could be accounted for by this pathway, suggesting that the remaining molecules must be retrieved from the apical surface before degradation.

Animals↗
PubMed1990

Synaptic localization and neural regulation of an N-acetylgalactosaminyl transferase in skeletal muscle.

We have previously documented the properties of a approximately 220-kDa cell surface glycosyltransferase that transfers N-acetylgalactosamine to oligosaccharide chains (Balsamo et al., 1986b). Because N-acetylgalactosamine-terminated carbohydrates are concentrated at the neuromuscular junction (Scott et al., 1988), we assayed skeletal muscle for the presence of the N-acetylgalactosaminyl transferase. Using immunohistochemical methods, we found that the enzyme is localized at neuromuscular junctions on normal adult rat muscle fibers. Biochemical assays confirm that junctional areas are highly enriched in the approximately 220-kDa immunoreactive species as well as in enzyme activity associated with the approximately 220-kDa species. This restricted distribution is dependent on synaptic integrity, as the enzyme appears extrasynaptically on denervation. These results provide a plausible metabolic basis for the localization of a synapse-specific carbohydrate and demonstrate that the expression of a glycosyltransferase is regulated by synaptic interactions.

Animals↗
PubMed1988

Purification and characterization of a bindable form of mitochondrial bound hexokinase from the highly glycolytic AS-30D rat hepatoma cell line.

Recent studies from this laboratory have demonstrated that a form of hexokinase characteristic of rapidly growing, highly glycolytic tumor cells is bound to an outer mitochondrial membrane receptor complex containing a Mr 35,000 pore protein (D. M. Parry and P. L. Pedersen, J. Biol. Chem., 258: 10904-10912, 1983; R. A. Nakashima, et al., Biochemistry, 25: 1015-1021, 1986). In new studies reported here the specificity of this receptor complex for binding hexokinase is defined, and a purification scheme is described which leads to a homogeneous and bindable form of the tumor hexokinase. In the AS-30D hepatoma, hexokinase activity is elevated more than 100-fold relative to liver tissue. The relative increase in hexokinase activity is 8 times greater than that of any other glycolytic enzyme. Hexokinase is the only glycolytic enzyme of AS-30D cells to exhibit a mitochondrial/cytoplasmic specific activity ratio greater than 1, showing a 3.5-fold elevation in the mitochondrial fraction. Purification of hexokinase is accomplished by preferential solubilization of the mitochondrial bound enzyme with glucose-6-phosphate, followed by high-performance liquid chromatography on gel permeation and anion exchange columns. The final fraction has a specific activity of 144 units per mg of protein, with a Km for glucose of 0.13 mM and for ATP of 1.4 mM. The purified tumor enzyme migrates as a single species upon sodium dodecyl sulfate: polyacrylamide gel electrophoresis with an apparent molecular weight of 98,000. Significantly, the purified tumor enzyme retains its activity for mitochondrial binding. Additional results derived from chromatographic, polyclonal antibody, and amino acid analysis studies indicate that the predominant rat hepatoma hexokinase species is related most closely to isozymic form(s) of the enzyme commonly referred to as type II, and least related to the liver type IV isozyme (glucokinase).

Amino Acids↗
PubMed1988

[Regulation of synaptogenesis in the adult skeletal muscle].

Several behaviors that axons exhibit during reinnervation of adult skeletal muscle demonstrate that they are guided by cues that the muscle provides. For example, axons form synapses on denervated but not on normally innervated muscle fibers; axons selectively reinnervate original synaptic sites on denervated muscle fibers; regenerating axons become specialized for synaptic transmission only in regions where they contact muscle fibers; and motor axons prefer to reinnervate muscles derived from matching levels of the body's rostro-caudal axis. This chapter describes these phenomena and summarizes progress toward identifying the soluble, membrane-bound, and extracellular matrix molecules that underly them.

Animals↗
PubMed1988

A synapse-specific carbohydrate at the neuromuscular junction: association with both acetylcholinesterase and a glycolipid.

With the aim of investigating the roles of carbohydrates in synapse formation, we have characterized a synapse-specific saccharide at the vertebrate neuromuscular junction. Two lectins of similar specificity (Dolichos biflorus agglutinin, DBA, and Vicia villosa-B4 agglutinin, VVA-B4) stain synaptic but not extrasynaptic regions of the rat muscle fiber surface and thus define a synapse-specific carbohydrate. Using these and other probes, we show that the carbohydrate moiety concentrated at the neuromuscular junction resembles N-acetylgalactosamine (GalNAc) linked in the beta-anomeric form to the termini of oligosaccharides. VVA-B4 also selectively stains neuromuscular junctions in human, mouse, rabbit, guinea pig, chick, frog, axolotl, snake, fish, and lamprey muscles, a phylogenetic conservatism that suggests a synapse-related role for GalNAc beta-terminal saccharides. AChE from muscle binds to DBA- and VVA-B4-agarose, and is thereby identified as a glycoconjugate bearing the synapse-specific carbohydrate. Assay of AChE isoforms reveals that asymmetric, collagen-tailed forms, known to be highly concentrated at the rat neuromuscular junction, bind DBA and VVA-B4, while globular forms, which are more widely distributed, do not. A second class of GalNAc-bearing glycoconjugates is demonstrable immunohistochemically with monoclonal antibodies to stage-specific embryonic antigen (SSEA)-3 (Shevinsky et al., 1982) and GM2 (Natoli et al., 1986), which recognize GalNAc-bearing glycolipids. These antibodies selectively stain neuromuscular junctions, where they recognize glycolipid-like molecules that bind VVA-B4 but are distinguishable from AChE. The association of a synapse-specific carbohydrate with at least 2 different synapse-specific molecules raises the possibility that the former plays a role in determining a property that the latter share, such as concentration at the synapse.

Acetylcholinesterase↗
PubMed1987

Stimulation of smooth muscle cell glycosaminoglycan synthesis by cultured endothelial cells is dependent on endothelial cell density.

Smooth muscle cells (SMC) cultured in the presence of endothelial cells (EC), or in EC-conditioned medium, show increased synthesis of glycosaminoglycans (GAG). We have found that both the amount and type of GAG produced by the SMC are dependent on the density of the EC. EC (porcine) at a low density (0.1-0.5 X 10(6) cells/25 cm2), or their conditioned media, where the most active per cell in stimulating GAG. All GAG were stimulated but the increase was due mostly to hyaluronic acid (HA). At intermediate densities (1.0 X 10(6)/25 cm2) stimulation was markedly reduced, but still present, and both HA and sulphated GAG were similarly increased. At high densities (1.5-3 X 10(6)/25 cm2) where EC were confluent there was very little stimulation of HA but continued stimulation of sulphated GAG synthesis. The shift in stimulation from HA to sulphated GAG with increasing density was most clearly demonstrated by the decrease in the HA to the chondroitin sulphate ratio. These findings provide support for the general concept that SMC metabolism may be affected by changes in the state of the endothelium.

Animals↗
PubMed1985

Effects of endothelial removal and regeneration on smooth muscle glycosaminoglycan synthesis and growth in rat carotid artery in organ culture.

Segments of rat carotid artery were maintained in serum-free and serum-supplemented media with endothelium both present and substantially removed by air drying. At intervals of 3, 7, and 14 days the synthesis of glycosaminoglycan across the vessel walls was determined by autoradiographic detection of incorporated [3H]glucosamine. In control carotids the typical pattern of incorporation was 40% of label in the intima, consisting of endothelium and subendothelial matrix, 23, 13, and 15% in the three medial layers (M1, M2, M3, respectively), and 9% in the adventitia. During the first week in culture the proportion, and often the amount, of label in M1 increased significantly. Following air drying labeling decreased markedly in M1 but often increased in M2 and M3. By 14 days residual endothelial cells had regenerated, and the pattern of incorporation in the medial layers beneath this new endothelium was the same as for the controls with a high level of labeling in M1. In areas free of endothelium incorporation in M1 remained at a low level. Digestion with chondroitinase ABC and Streptomyces hyaluronidase showed that the changes in M1-labeling levels were due to changes in the amounts of both hyaluronic acid and sulfated glycosaminoglycan, whereas pulse and continuous labeling studies showed that the different labeling levels for the various layers and conditions were due to different rates of synthesis and not degradation. Carotids were also labeled with [3H]thymidine. Control and regenerating endothelia were active in serum-free and serum-supplemented media and had similar mitotic indices. Indices for smooth muscle cells in M1, however, were generally very low and were not affected by the presence or absence of endothelium. We conclude that endothelial removal results in decreased glycosaminoglycan synthesis in the inner media, that mitotically active endothelium correlates with increased glycosaminoglycan synthesis in the inner media, and that these changes occur independently of smooth muscle cell growth.

Animals↗
PubMedDate not supplied

Fluoroprobe quantification of viable and non-viable cells in human coronary and internal thoracic arteries sampled at autopsy.

Viable and non-viable cells in coronary and internal thoracic arteries, collected at autopsy 7-24 h post-mortem from individuals 15-81 years of age, were detected using the fixable fluoroprobes 5-chloromethylfluorescein diacetate (green) and ethidium homodimer-1 (orange/red). Viability status of individual endothelial and smooth muscle cells was confirmed by simultaneous autoradiographic detection of incorporated [3H]glucosamine. Twenty-five percent of coronary and 42% of internal thoracic arteries contained viable cells up to 24 h following death. For the majority of viable vessels the mean percentage of viable cells ranged between 60 and 80% with no significant difference between coronary and internal thoracic arteries and no relationship with either age of the donor or with time to autopsy. Non-viable cells were usually distributed fairly evenly amongst viable cells but this pattern could not be assumed. In a number of vessels non-viable cells were variably clustered in different regions of vessel wall. These findings confirm that vessels sampled at autopsy can be used for metabolic studies with the caveat that assessment of cell viability is a necessary prerequisite for interpretation of results.

Arteries↗