Eric M. Carey

Changes in the activities of de novo fatty acid synthesis and palmitoyl-CoA synthetase in relation to myelination in rabbit brain

1. Age-related changes in the activities of fatty acid synthetase and palmitoyl-CoA synthetase (EC 6.2.1.3) have been determined in rabbit brain from the foetal stage through to maturity. 2. Fatty acid synthetase was most active in the soluble fraction of brain homogenates at 5 days of age, prior to the active phase of myelination. Palmitic acid was the major fatty acid synthesised throughout development. 3. Palmitoyl-CoA synthetase had a constant specific activity in the full homogenate, but in the microsomal fraction reached a maximum specific activity at 15-20 days of age. The specific activity was higher than in the mitochondrial fraction which declined from birth. Most of the palmitoyl-CoA synthetase activity was present in the fraction containing cell membranes plus nuclei. 4. From a comparison of the total activities of the enzymes involved in the metabolism of fatty acids in the brain, de novo fatty acid synthesis may be rate limiting compared with esterification of synthesised fatty acids, but not in the further transformations of the synthesised fatty acids.

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U‐14C]glucose and [1‐14C]acetate, with lipid synthesis from exogenous albumin‐bound [1‐14C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain‐length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non‐esterified fatty acid pool in the two situations. At the period of their most active formation, the very long‐chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C18 acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.

Changes in the lipid and fatty acid composition of developing rabbit brain

Abstract 1. 1. In the developing rabbit brain there is a progressive accumulation of most lipids particularly cholesterol and sphingolipids as in the brain of other animals. The highest rate of accumulation corresponded to the early period of myelination in the rabbit. 2. 2. Phospholipids (with the exception of phosphatidylethanolamine) are not accumulated to the same extent as other lipids. 3. 3. Each lipid analysed showed a characteristic fatty acid pattern. Cerebrosides showed the greatest changes in composition during maturation and together with sphingomyelin contained most of the very long-chain fatty acids characteristic of these lipids in the myelin membrane. 4. 4. Fatty acids of dietary lipid of the neonate are incorporated only to a slight extent into brain lipids, mainly as triglycerides.

Review: Diagnostic and therapeutic applications of rat basophilic leukemia cells.

The research into understanding of the immunological processes is often difficult due to several factors complicating the isolation and culturing of primary degranulating cells like mast cells and basophils. The establishment of rat basophilic leukemia (RBL) cell line as an efficient and reliable experimental research tool was considered a major advance toward the understanding of the wild-type mast cell populations biology. The development of sub-clone RBL-IV (HR+) led to the isolation of histamine-secreting RBL-2H3 cell line. Since then, RBL-2H3 cells have been extensively used for studying the IgE high affinity receptor (FcɛRI) interactions with their ligand, the IgE antibody. This cell line has been employed for generating human and more recently canine and equine FcɛRIα-transfected RBL cell lines facilitating an assessment of the residues involved in the complementary interaction between the IgE molecules from these species and their cognate high affinity receptor. A proteomics-based approach to the definition of IgE-receptor-mediated signaling pathways was also carried out using this cell line. Furthermore, RBL-2H3 cells have the potential of being used to assess the potential allergenicity of antigens to humans and other animals like dogs and horses which are known to suffer from similar allergic manifestations.

UDP-Galactose:Ceramide Galactosyl Transferase of Isolated Oligodendroglia

Abstract: The activity of UDP‐galactose:ceramide galactosyl transferase (CGalT) has been studied in isolated oligodendroglia from bovine brain white matter and myelinating rat brain. The specific activity and activity per mg DNA are 4‐ and 10‐fold higher in rat oligodendroglia compared with neuronal perikarya from rat brain, and is higher in oligodendroglia from myelinating rat brain compared with bovine oligodendroglia. In membranes isolated from oligodendroglia, the specific activity decreased in the order endoplasmic reticulum > plasma membrane > myelin.

Fatty acid metabolism in the microsomal fraction of developing rabbit brain.

1. Incorporation of [14C]malonyl-CoA into fatty acid and lipid by the microsomal fraction from developing rabbit brain has been determined. 2. The specific activity for malonyl-CoA incorporation into preformed fatty acid reached a maximum at 15-20 days of age and the increase in enzyme activity paralleled that of palmitoyl-CoA synthesis in the microsomal fraction. 3. Most of the preformed fatty acid was derived from microsomal membrane lipid, and added acyl-CoA only slightly increased the incorporation. Inhibition occurred at concentrations in excess of 2 muM acyl-CoA. Endogenous acyl-CoA may be formed by acyl-group turnover through the action of phospholipase A and acyl-CoA synthetase. 4. Added 14C-labelled acyl-CoA was elongated to a longer chain-length product than endogenous fatty acid elongated with [14C]malonyl-CoA. 5. Both ATP and acyl-CoA influence the incorporation of elongated fatty acids into complex lipids, possibly through their effect on acyl-CoA synthetase.

Phospholipid exchange activity in developing rat brain.

Phospholipid exchange activity has been determined in the supernatant fraction of rat brain from birth through to maturity by measuring the protein-catalysed transfer of total and individual 32P-labelled phospholipids from microsomal membranes to mitochondria, and the transfer of [14C]phosphatidylcholine from liposomes to mitochondria. Transfer activity has also been compared in brain and liver supernatant. Overall phospholipid exchange activity in the brain increased only slightly with age. The activity at birth was 75% of the adult value. However, the transfer of individual phospholipids showed markedly different trends during postnatal brain development. The transfer of phosphatidylinositol (PI) and ethanolamine phospholipids increased postnatally to a maximum at 9 days of age, with lowest values in adult brain. Phosphatidylcholine (PC) transfer increased from 9 days to reach maximum values in the mature brain. The transfer of sphingomyelin was highest immediately after birth. PI transfer activity was higher in brain than liver, while PC and ethanolamine phospholipid transfer activity was higher in liver. The heterogeneity of phospholipid exchange proteins in central nervous system tissue is reflected in the developmental changes in exchange activity towards individual phospholipids. The various exchange proteins appear to have separate induction mechanisms. The presence of exchange-protein activity from birth in the rat indicates the functional importance of phospholipid transport during cell acquisition and membrane proliferation. Activity is not primarily associated with membrane formation such as the formation of the myelin sheath, and therefore is more likely to be involved in the process of phospholipid turnover.

Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines

The expression of several isoforms of the small-molecular-weight Rab3 GTP-binding proteins is a characteristic feature of all cell types undergoing regulated exocytosis, in which Rab3 proteins are considered to regulate the assembly/disassembly of a fusion complex between granule and plasma membrane in a positive and negative manner through interaction with effector proteins. The pattern of Rab3 protein expression may, therefore, provide a subtle means of regulating exocytosis. To investigate the relationship between Rab3 expression and secretory activity, we assessed the differential expression of individual Rab3 proteins in high- and low-secreting clones of the rat basophilic (RBL) cell line. mRNAs for Rab3 isoforms (a-d) were analyzed by constructing cDNA libraries of high- and low-secreting RBL clones. The relative abundance of mRNAs for Rab3 isoforms was initially determined from the clonal frequency of corresponding cDNA clones. RT-PCR using isoform-specific primers was successfully applied to the quantitation of Rab3a mRNA. The presence of individual Rab3 proteins was revealed by SDS-PAGE and immunoblotting, and also by in situ immunofluorescence confocal microscopy. We present evidence that Rab3a and Rab3c are expressed at high levels in the low-secreting variant, while Rab3d is predominant in the high secretor. Levels of the Rab3 effector proteins, Rabphilin and Noc2, are similar in both RBL cell lines. Subcellular fractionation of unstimulated high and low secretor RBL clones revealed that in both cell types Rab3a has a cytoplasmic location while Rab3d is present in a membrane/organelle fraction containing secretory vesicles. Differences in the pattern of expression of Rab3 isoforms in the two RBL cell lines and their localisation may influence the secretory potential. Furthermore, the presence of Rab3 and effector proteins indicates that the mechanism for regulated exocytosis in cells of mast cells/basophil lineage appears similar to that in pre-synaptic vesicles and pancreatic beta-cells.

Comparison of the acylation of sn-glycerol 3-phosphate and membrane-bound lipid in the microsomal fraction from rabbit brain throughout maturation

1. Age-related changes in the specific activity of palmitoyl-CoA synthetase, sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and the esterification of [3H]palmitate into endogenous lipid in the microsomal fraction from rabbit brain have been determined throughout development. 2. The increased specific activity of sn-glycerol 3-phosphate acyltransferase at the onset of myelination (rising in parallel with other lipogenic enzymes) is consistent with a direct role of the acyltransferase in promoting the accumulation of cerebral lipid. In adult brain microsomes, although the specific activity was low, the total activity was only 20% lower than during active myelination. 3. Palmitoyl-CoA, synthesized by the palmitoyl-CoA synthetase in the microsomal membrane, was the preferred substrate for the esterification of sn-glycerol 3-phosphate. There was no evidence for a pool of palmitoyl-CoA formed from palmitate. 4. The esterification of [3H]palmitate into membrane-bound lipid remained high throughout development and may be part of an acyl-exchange cycle via lysophospholipids. [3H]palmitate was incorporated into both neutral lipids and phospholipids, while phosphatidic acid was the major product of sn-[1(3)-3H]-glycerol-3-phosphate esterification. 5. The microsomal fraction contained a pool of unesterified fatty acid, which was activated and esterified into sn-glycerol 3-phosphate.

Fatty acid biosynthesis. X. Specificity for chain-termination of fatty acid biosynthesis in cell-free extracts of lactating rabbit mammary gland.

Abstract 1. 1. The pattern of fatty acids synthesised from [1−14C]acetate by subcellular fractions of lactating rabbit mammary gland fortified by appropriate cofactors was found to be dependent on the concentration of the fractions as measured by protein. 2. 2. At high concentrations (14–22 mg protein/ml), the cell-free homogenate, the microsomal plus particle-free supernatant, and the particle-free supernatant fractions all synthesised a high proportion of the medium-chain fatty acids (C8:0) and C10:0) which are characteristic of rabbit milk. 3. 3. Dilution of these fractions to less than 1 mg protein/ml caused a progressive increase in the proportion of long-chain acids (C14:0 and C16:0) synthesised. 4. 4. With concentrated subcellular fractions, synthesis of medium-chain fatty acids was not dependent on the rate of fatty acid synthesis, the concentration of malonyl-CoA, or the amount of fatty acid synthetase present. 5. 5. Though there was a correlation between the synthesis of medium-chain fatty acids and of triglyceride by particulate fractions, esterification of synthesised fatty acids did not influence the proportion of medium-chain acids formed. 6. 6. Evidence is presented that chain termination at C8:0 and C10:0 acids is controlled by a factor (or factors) present in the particle-free supernatant fraction. This factor is not lost on dialysis, and can be precipitated between 40 and 100% saturation with (NH4)2SO4 or by heating.