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Biomedical subjects

R M Gould

Publications and source records attributed to R M Gould.

At least 55 records · Page 3Linked to original sources
PubMed1985

Axonal transport of polyamines in intact and regenerating axons of the rat sciatic nerve.

The axonal transport of putrescine or its polyamine derivatives spermidine or spermine is a subject of some debate. We investigated this question by injecting [3H]putrescine into the lumbar spinal cord of the rat and measuring the accumulation of radioactivity central to ligatures placed on intact and regenerating sciatic nerves. In normal nerves, approximately twice as much radioactivity built up proximal to these ligatures 2 or 3 days after injection than at more distal ligatures used to control for accumulation of radioactivity which might be due to tissue damage alone. In regenerating nerves the amount of radioactivity accumulating at the ligature was approximately five times that at the distal ligature and two to three times greater than in intact nerves. The identity of the radioactivity in regenerating nerves, determined on an amino acid analyzer, was found to be primarily spermidine and an unknown compound that migrated as a frontal elution peak. Autoradiographic analysis showed that the radioactivity was largely confined to axons, but a significant amount of the silver grains was associated with Schwann cells and myelin sheaths surrounding labeled axons in both intact and regenerating nerves. The data indicate that polyamine derivatives of putrescine are transported axonally in rat sciatic nerves, and some of this transported material accumulates in Schwann cells surrounding the labeled axons. These processes are apparently augmented during regeneration of the injured axons.

Animals↗
PubMed1985

Effect of electrical stimulation on phosphoinositide metabolism in rat sciatic nerve in vivo.

The metabolism of phosphoinositides in rat sciatic nerves in vivo during electrical stimulation was studied. Nerves were prelabeled by injection of [2-3H]-myo-inositol alone for periods of 2 and 20 h or together with [32P]orthophosphate for 2 h and then electrically stimulated (100 Hz) for 5 or 20 min. Contralateral unstimulated nerve served as the control. When tritiated myo-inositol was used alone for prelabeling the nerves, approximately 6% and 14% of the label was incorporated into lipids after 2 h and 20 h, respectively. Both 5 and 20 min of electrical stimulation caused an insignificant change in the percentage of radioactivity recovered in lipids from the nerves prelabeled with either myo-inositol or with a mixture of myo-inositol and phosphate. The proportion of label associated with phosphoinositides of nerves prelabeled with myo-inositol for both 2 h and 20 h showed an increase in phosphatidyl-inositol-4-phosphate at the expense of phosphatidylinositol in stimulated nerves. Similar results were obtained with nerves prelabeled for 2 h with a mixture of [32P]orthophosphate and [2-3H]myo-inositol. No significant changes in the radioactivity associated with water-soluble inositol phosphates were found in stimulated versus control nerves.

Animals↗
PubMed1983

Autoradiographic and fine structural study of chitin deposition in the cuticle of a barnacle using [3H]-D-glucosamine incorporation.

[3H]-D-Glucosamine was injected into the rostral sinus of Balanus eburneus (barnacle) and the distribution of labelled chitin in the cuticle was studied with autoradiography and electron microscopy. When the pattern of labelling was examined in different body regions of the same organism where thickness of fully formed cuticle varied, it was observed that the rate of chitin deposition varied, being greater in thick than in thin regions. The density of Ag grains overlying cuticle was also greater in the thick regions. When the pattern of labelling was examined in regions of cuticle, comparable in thickness, taken from a series of organisms sacrificed at different time points a comparable value for the rate of chitin deposition was obtained. In addition, asynchrony in deposition of cuticle in different body regions of the same organism as well as uptake of the label by substances other than chitin, i.e. glycogen and glycoproteins were described.

Animals↗
PubMed1983

Phospholipid synthesis in the squid giant axon: incorporation of lipid precursors.

The squid giant axon and extruded axoplasm from the giant axon were used to study the capacity of axoplasm for phospholipid synthesis. Extruded axoplasm, suspended in chemically defined media, catalyzed the synthesis of phospholipids from all of the precursors tested. 32P-Labeled inorganic phosphate and gamma-labeled ATP were actively incorporated into phosphatidylinositol phosphate, while [2-3H]myo-inositol and L-[3H(G)]serine were actively incorporated into phosphatidylinositol and phosphatidylserine, respectively. Though less well utilized. [2-3H]glycerol was incorporated into phosphatidic acid, phosphatidylinositol, and triglyceride, and methyl-3H]choline and [1-3H]ethanolamine were incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Isolated squid giant axons were incubated in artificial seawater containing the above precursors. The axoplasm was extruded following the incubations. Although most of the product lipids were recovered in the sheath (composed of cortical axoplasm, axolemma, and surrounding satellite cells), significant amounts (4-20%) were present in the extruded axoplasm. With tritiated choline and myo-inositol, the major labeled phospholipids found in both the extruded axoplasm and the sheath were phosphatidylcholine and phosphatidylinositol, respectively. With both glycerol and phosphate, phosphatidylethanolamine was a major labeled lipid in both axoplasm and sheath. These findings demonstrate that all classes of phospholipids are formed by endogenous synthetic enzymes in axoplasm. In addition, we feel that the different patterns of incorporation by intact axons and extruded axoplasm indicate that surrounding sheath cells contribute lipids to axoplasm. A comprehensive picture of axonal lipid metabolism should include axoplasmic synthesis and glial-axon transfer as pathways complementing the axonal transport of perikaryally formed lipids.

Animals↗
PubMed1983

Phospholipid synthesis in the squid giant axon: enzymes of phosphatidylinositol metabolism.

We examined the properties of several enzymes of phospholipid metabolism in axoplasm extruded from squid giant axons. The following synthetic enzymes, CDP-diglyceride: inositol transferase (EC 2.7.8.11), ATP:diglyceride phosphotransferase, diglyceride kinase (EC 2.7.2.-), and phosphatidylinositol kinase (EC 2.7.1.67), were all present in axoplasm. Phospholipid exchange proteins, which catalyzed the transfer of phosphatidylinositol and phosphatidylcholine between membrane preparations and unilamellar lipid vesicles, were also found. However, we did not find conditions under which the synthesis of CDP-diglyceride, phosphatidylserine, and phosphatidylinositol-4,5-diphosphate could be measured. Subcellular fractionation by differential centrifugation showed that the axoplasmic inositol transferase and phosphatidylinositol kinase activities were largely "microsomal," while the diglyceride kinase and exchange protein activities were primarily "cytosolic."

1-Phosphatidylinositol 4-Kinase↗
PubMed1981

4S RNA is transported axonally in normal and regenerating axons of the sciatic nerves of rats.

Experiments were designed to determine if following injection of [3H]uridine into the lumbar spinal cord of the rat, [3H]RNA could be demonstrated within axons of the sciatic nerve, and if 4S RNA is the predominant RNA species present in these axons. In one experiment the left sciatic nerve of a rat was crushed. Two days later 170 microCi of [3H]uridine was injected into the vicinity of the lumbar ventral horn cells. Ten days after injection, rats were sacrificed and sciatic nerves were prepared for autoradiography. Photomicrographs were taken of labeled areas of intact and regenerating nerves and grains were counted over Schwann cells, myelin, axons and other unspecified areas. In both intact and regenerating sciatic nerves more than 20% of the silver grains were associated with motor axons and approximately 40% were found over cytoplasm of Schwann cells surrounding these axons. These data indicate an intra-axonal localization of RNA in sciatic nerve axons, as well as an active transfer of RNA precursors from axons to their surrounding Schwann cels. In separate studies, the left sciatic nerve was crushed and 10 days later [3H]uridine was bilaterally injected intraspinally into 6 rats. Four control rats were sacrificed at 14 or 20 days after injection. In the remaining 2 rats the sciatic nerve was cut 14 days after injection and the distal part of the nerve was allowed to degenerate for 6 days before sacrificing the rat. Thus, the distal portion of the nerve contained Schwann cells labeled by axonal transport but lacked intact axons. RNA was isolated from experimental and control nerve segments by hot phenol extraction and ethanol precipitation. RNA species (28S, 18S and 4S) were separated by polyacrylamide gel electrophoresis and radioactivity was measured in a liquid scintillation counter. Control groups had RNA profiles similar to those already described, with greater than 30% of the radioactivity present as 4S RNA. The proximal portions of nerve taken from the group in which nerves were cut, had a similar amount of radioactivity present as 4S RNA. However, in the distal segments of these nerves (in which the axons had degenerated thus creating an 'axon-less' nerve) the amount of radioactivity in the 4S peak decreased to approximately 15% of the total RNA, suggesting that 4S RNA is the predominant if not the only RNA present in these axons. These results strongly indicate that both intact and regenerating sciatic nerves of rats selectively transport 4S RNA along their motor axons.

Animals↗
PubMed1981

Internodal distribution of phosphatidylcholine biosynthetic activity in teased peripheral nerve fibres: an autoradiographic study.

Radioactive choline injected into mouse sciatic nerve is rapidly incorporated into into phosphatidylcholine. Sites of deposition of this phospholipid have been localized along the internode in autoradiography prepared from individually teased fibres. The newly synthesized lecithin formed during 20 min or 2 h labelling periods is concentrated in the perinuclear region of the Schwann cell and in strands radiating from this portion of the cell. This labelling pattern, representing a complex of enzyme activities, is distributed in a similar, though not identical, fashion to that of Schwann cell mitochondria as localized by histochemical methods. These findings suggest that soluble and membrane-associated enzymes required for phosphatidylcholine formation are distributed in Schwann cell cytoplasm along superficial longitudinally oriented channels as depicted in recent freeze-fracture studies.

Animals↗
PubMed1980

Enzymes of phospholipid synthesis: axonal versus Schwann cell distribution.

Using quantitative EM autoradiography to localize sites of incorporation of tritiated inositol and choline into mouse sciatic nerve, we observed a substantial axon-based phosphatidylinositol synthesis, but no axonal phosphatidylcholine synthesis. In the present communication we provide biochemical evidence for the axonal transport of CDP-diglyceride:inositol transferase (EC 2.7.8.11), the terminal enzyme in de novo phosphatidylinositol biosynthesis. Axonal transport of 1,2-diacyl-glycerol:CDP-choline choline phosphotransferase (EC 2.7.8.2), required for de novo phosphatidylcholine synthesis, was not apparent in these studies. During subcellular fractionation activities for the synthesis of phosphatidylinositol by inositol transferase (IT) and phosphatidylcholine by choline phosphotransferase (CPT) were recovered in crude microsomal fraction of rat sciatic nerve. However, CPT was much more highly enriched in the microsome fraction than IT, which may be an indication of the different subcellular localizations of these enzymes. Following ligation, we detected localized increases in the activities of both enzymes in 5 (and 3) mm segments taken immediately proximal and distal to the ligature. Both activities increased in a linear fashion in the proximal segments over the ensuing 72 h period. It took about 40 h (IT) and 56 h (CPT) for the activities in the segments proximal to the ligature to double compared to unligated contralateral (control) nerves. The time-dependent accumulation of IT was primarily due to axonal transport, while that of CPT was largely a result of increased enzyme activity in local Schwann cells. Evidence came from double ligation studies, where a proximal ligature, acting to restrict orthograde axonal transport, reduced accumulation in a distal ligature by 80% for IT, but only 28% for CPT. Conversely, blockage of the Schwann cell response with actinomycin D, reduced accumulation of CPT by 83% and IT by only 36%. Finally, light microscopic autoradiography was used to show that in the segment proximal to the ligature, tritiated inositol incorporation into lipid was primarily axonal, whereas that of tritiated choline remained primarily associated with Schwann cells.

Animals↗
PubMed1977

Incorporation of glycoproteins into peripheral nerve myelin.

Peripheral nerve myelin contains a dominant low molecular weight glycoprotein called Po. To study the metabolism of this glycoprotein, tritiated fucose was injected into the peripheral nerves of adult mice and developing rats, and the temporal distribution of label was examined by autoradiography and gel electrophoresis. Mice and rat pups, injected with fucose, were sacrificed from 1 h to 98 days later. Series of autoradiographs were prepared. At the shortest labeling periods, newly formed product was confined to juxtanuclear Schwann cell cytoplasm, in association with regions rich in Golgi apparatus. After longer labeling periods, silver grain levels in Schwann cell cytoplasm decreased; concomitantly, there was an increase of silver grains associated with myelin. In adult animals, label associated with myelin was concentrated over outer layers of thickly myelinated fibers. Even at the longest time intervals examined (72 and 98 days), this distribution of label was largely retained. In contrast, in developing animals, label became associated with inner layers of the thicker sheaths. At no time was label observed over axons. Gel electrophoresis revealed that tritiated fucose was a suitable precursor for the faster migrating peripheral nerve glycoprotein(s). At all times examined, there was a single major peak of radioactivity that co-migrated on sodium dodecyl sulfate (SDS) acrylamide gels with the Po protein. Sometimes, a faster migrating shoulder of radioactivity was noted. With increased labeling periods, there was an enrichment of radioactivity associated with Po, indicative of a relatively slow turnover rate.

Animals↗
PubMed1976

Incorporation of newly formed lecithin into peripheral nerve myelin.

Radioactive choline was used to study the metabolism and movement of choline-containing phospholipids in peripheral nerve myelin of adult mice. Incorporation at various times after intraperitoneal injection was measured in serial segments of sciatic nerve as well as in myelin isolated from those segments. At no time (1 h to 35 days) could a proximal-distal difference in the extent of labeling be demonstrated. This finding suggests that incorporation of precursor choline phospholipids into nerve membranes is a local event, with little contribution from the neuronal perikaryon via axoplasmic transport. Autoradiographic investigations were undertaken to elucidate the pattern of movement of radioactive choline-labeled phospholipids, predominantly lecithin, into the myelin sheaths of the sciatic nerve. A sequence of autoradiographs was prepared from animals sacrificed between 20 min and 35 days after a microinjection of precursor directly into the nerve. Analysis of these autoradiograms revealed that labeling is initially concentrated in the Schwann cell cytoplasm. Later, the label moves first into the outer regions of the myelin sheaths and is eventually distributed evenly throughout the inner and outer layers of the sheath. At no time is there a build-up of label in the axon. The rate of uptake of precursor and subsequent redistribution of lecithin into the myelin were also examined in frog sciatic nerve (18 degrees C). Both uptake and redistribution processes were considerably slower in the cold-blooded animal.

Animals↗
PubMed1970

Metabolism of Phosphatidylglycerol and Lysyl Phosphatidylglycerol in Staphylococcus aureus.

The metabolism of phosphatidylglycerol and lysyl phosphatidylglycerol was studied in Staphylococcus aureus under four conditions: growing at pH 7.0 and 5.2, and not growing (resting) at pH 7.0 and 5.2. Measurements of the amounts of phosphatidylglycerol and lysyl phosphatidylglycerol, as well as labeling and pulsechase experiments, revealed that the phosphate group of the former and the lysyl group of the latter were in a state of active turnover. A marked decline in the cellular level of phosphatidylglycerol observed when cells were resting at pH 5.2 was found to be caused by both a decrease in synthesis and an increase in catabolism. The level of lysyl phosphatidylglycerol was found to be relatively constant under the four incubation conditions, although the lysyl moiety was in a state of turnover. Experiments designed to test the possible role of lysyl phosphatidylglycerol as a lysyl group donor in biosynthetic processes or in lysine transport were negative; no evidence to support the hypothesis that lysyl phosphatidylglycerol serves as an intermediate was obtained.

Journal Article↗