Influence of intron length on alternative splicing of CD44.

Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

v-Myb can transform and regulate the differentiation of melanocyte precursors.

The activity of the c-Myb transcription factor is essential for the development of definitive multi- and uni-lineage progenitors of the haemopoietic system. Reflecting this requirement, c-Myb has been oncogenically activated by transduction in the E26 avian retrovirus which elicits an acute leukaemia by transforming haemopoietic progenitors. Here, we report the novel finding that Myb in cooperation with EGF receptor signalling can be used to generate clonally expanded populations of transformed cells which have the phenotype of melanocyte precursors. Through the use of a conditional temperature sensitive mutant of Myb, we show that in the transformed cells Myb regulates commitment to melanocyte differentiation and possibly proliferation. These results add to our understanding of the roles of c-Myb beyond the haemopoietic system and to our knowledge and means of investigating the importance of transcription factors in the melanocyte lineage.

Effects of orthovanadate on salt and water effluxes from the gills of seawater eels, Anguilla anguilla.

Orthovanadate (5 . 10(-7) M) perfused through isolated gills at a constant rate increased the perfusion pressure by 40% but inhibited the effluxes of Na+ and Cl- by 40%. Water efflux was unaltered. Ouabain (10(-4) M) and rotenone (10(-4) M) influenced salt and water effluxes in the same way but did not alter perfusion pressures. Orthovanadate (10(-5) M) perfused at constant rate increased the pressure nearly 2.5-fold; under these conditions effluxes of Na+, Cl- and H2O were all increased approximately 2.5-fold.

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n-3), to eicosapentaenoic acid, 20:5(n-3), in a cell line from the turbot, Scophthalmus maximus.

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C18 to C20 polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n-3), and its elongation product, 20:4(n-3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 microM), U-14C (1 microM) or deuterated (d5; 25 microM) fatty acids. Stearidonic acid, 18:4(n-3), was metabolised to 20:5(n-3) in both cells lines, but more so in AS than in TF cells. Delta5 desaturation was more active in TF cells than in AS cells, whereas C18 to C20 elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n-3)) were produced by both cell lines, although there was significant production of 22:5(n-3) in both cultures, especially when 20:4(n-3) was supplemented. We conclude that limited elongation of C18 to C20 fatty acids rather than limited fatty acyl Delta5 desaturation accounts for the limited rate of conversion of 18:3(n-3) to 20:5(n-3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C18 to C20 PUFA by the turbot and the Atlantic salmon in vivo.

Fine mapping of glycerol kinase deficiency and congenital adrenal hypoplasia within Xp21 on the short arm of the human X chromosome.

We have studied patients with Duchenne muscular dystrophy (DMD), DMD together with glycerol kinase (GK) deficiency, or DMD together with both GK deficiency and congenital adrenal hypoplasia (AHC). Analysis of deletions in these patients allows the mapping of these mutations in Xp21. The following order is proposed: Xpter - L1 - AHC - GK - DMD - Xcen. One of the boys with DMD, GK, and AHC is shown by pulsed-field-gel electrophoresis to have a deletion which has a proximal endpoint at least 500 kb distal from the pERT87 (DXS164) locus.

Linkage analysis in the fragile X syndrome using multiple distal Xq polymorphic DNA markers.

Linkage data using the polymorphic loci F9, DXS105, DXS98, DXS52, DXS15, and F8 and the DNA probe 1A1 are presented from 14 families segregating for fragile X [fra(X)] syndrome. Recombination fractions corresponding to the maximum LOD scores obtained by two-point linkage analysis suggest that DXS98 (Zmax = 3.23, theta = 0.0) and DXS105 (Zmax = 2.09, theta = 0.0) are the closest markers proximal to FRAXA and that DXS52 is the closest distal marker (Zmax = 3.55, theta = 0.16). FRAXA is located within a 25 cM interval between F9 and DXS52, coincident with DXS98, on multipoint linkage analysis. Phase-known three way crossover information places F8 outside the cluster (DXS52, DXS15, 1A1). Confidence limits for the markers DXS98 and DXS52 are relatively wide (0.0-0.15 and 0.06-0.31, respectively), but when used in combination with cytogenetic examination offer improved carrier detection in comparison with cytogenetic analysis alone.

Mapping of a cerebellar degeneration related protein and DXS304 around the fragile site.

We have localized the gene encoding a cerebellar degeneration related (CDR) protein to a region proximal to the fragile site close to DXS98 and DXS105. This gene is polymorphic with the enzyme RsaI and therefore also provides a new genetic marker in this region. We have refined the localization of the locus DXS304 distal to the breakpoint in a patient suffering from Hunter disease. This confirms the localization of DXS304 distal to the fragile site previously suggested by linkage studies and localizes the fragile X mutation to a relatively small region between the Hunter breakpoint and the breakpoint in another hybrid B17.

Nociceptin/orphanin FQ (N/OFQ) induces a quasi-morphine abstinence syndrome in the rat.

Nociceptin/orphanin FQ (N/OFQ) is a neuropeptide that exerts antiopiate effects under some circumstances, and there is evidence that it contributes to opiate tolerance. This raises the question, might N/OFQ also contribute to opiate dependence and abstinence? Twenty-two male Sprague-Dawley rats were cannulated in the third ventricle and challenged 7 days later by third ventricle injection of 50, 200 or 1,000 ng N/OFQ or saline alone. Each rat was observed under "blind" conditions for 30 min beginning 15 min after onset of the third ventricle injection. There was a significant positive linear trend of signs as a function of N/OFQ dose. Subjects receiving saline had 18.0+/-2.0 (mean+/-SEM) overall abstinence-like signs, whereas subjects receiving 50, 200 or 1000 ng N/OFQ had 35.2+/-3.6, 49.8+/-2.6 and 63.5+/-9.7 signs, respectively. In 16 additional rats, abstinence-like signs induced by 1000 ng N/OFQ were significantly attenuated by low SC doses of morphine or clonidine. These results raise the possibility that N/OFQ might contribute to opiate dependence and subsequent abstinence syndrome. On the other hand, N/OFQ over a wide dose range induced abstinence signs with similar potency in morphine dependent and non-dependent rats.

Fatty acid composition, eicosanoid production and permeability in skin tissues of rainbow trout (Oncorhynchus mykiss) fed a control or an essential fatty acid deficient diet.

Rainbow trout (Oncorhynchus mykiss) were fed either a control diet containing fish oil or an essential fatty acid (EFA) deficient diet containing only hydrogenated coconut oil and palmitic acid as lipid source (93.4% saturated fatty acids) for 14 weeks and the fatty acid compositions of individual phospholipid classes from skin and opercular membrane (OM) determined. The permeability of skin and OM to water and the production of eicosanoids in skin and gills challenged with the Ca2+ ionophore A23187 were also measured. Phospholipid (PL) fatty acid compositions were substantially modified in EFA-deficient fish, with increased saturated fatty acids and decreased polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) and eicosapentaenoic acid (EPA), while docosahexaenoic acid (DHA) was largely retained. The onset of EFA deficiency was shown by the appearance of n-9 PUFA, particularly 20:3n-9. The main effects of EFA deficiency on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were to increase saturated fatty acids and monoenes, especially 16:1 and 18:1, and to decrease EPA and DHA. The content of DHA in phosphatidylserine (PS) was high in control animals (40% in skin and 35% in opercular membrane) and was mostly retained in EFA deficient animals. Arachidonic acid (AA) was the most abundant PUFA esterified to phosphatidylinositol (PI) and was significantly reduced in EFA deficient animals (from 31% to 13% in skin), where a large amount of 20:3n-9 (9% in skin) was also present. Influxes and effluxes of water through skin and opercular membrane were measured in vitro. No differences were detected between rainbow trout fed the control or the EFA deficient diet. 12-Hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHE) could not be detected in skin from control or EFA deficient fish. There was no difference between control and EFA deficient trout in the levels of leukotriene C4 (LTC4) and leukotriene C5 (LTC5) in skin cells challenged with the calcium ionophore A23187, and of prostaglandin F2alpha (PGF2alpha), 12-HETE and 12-HEPE in gill cells challenged similarly. Prostaglandin F3alpha (PGF3alpha) production by ionophore stimulated gill cells was significantly reduced in fish fed the EFA-deficient diet. 14-HDHE produced by gill cells was 3.3 fold higher in EFA deficient fish compared to controls.

Molecular species composition of glycerophospholipids from white matter of human brain.

The molecular species composition of the major glycerophospholipids from white matter of human brain were determined by high-performance liquid chromatography of the 3,5-dinitrobenzoyl derivatives of the corresponding diradylglycerols. In phosphatidylcholine (PC) and phosphatidylserine (PS), molecular species containing only saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) comprised 85.7 and 82.4% of the respective totals, with 18:0/18:1 predominant in PS and 16:0/18:1 in PC. These molecular species were also abundant in phosphatidylethanolamine (PE), but in this phospholipid species containing polyunsaturated fatty acids (PUFA), largely 18:0/22:6n-3 and 18:0/20:4n-6, accounted for over half the total; 18:1/18:1 was also abundant in PE. In contrast, 1-O-alk-1'-enyl-2-acyl sn-glycero-3-phosphoethanolamine (GPE) had much more SFA- and MUFA-containing species, predominantly 16:0a/18:1, 18:0a/18:1 and 18:1a/18:1, with low amounts of species containing 20:4n-6 and 22:6n-3. In alkenylacyl GPE, 22:4n-6 was the major PUFA and 16:0a/22:4n-6 and 18:1a/22:4n-6 the main PUFA-containing species. There was six times more 22:6n-3, twice as much 20:4n-6 and half the amount of 22:4n-6 in PE as compared to alkenylacyl GPE.

1-O-alk-1'-enyl-2-acyl-glycerophosphoethanolamine content and molecular species composition in fish brain.

Ethanolamine glycerophospholipids from the brains of both trout and cod comprised 36-38% of 1-O-alk-1'-enyl-2-acyl-glycerophosphoethanolamine (GPE) determined using two methods. In 1-O-alk-1'-enyl-2-acyl-GPE from trout brain, the main molecular species were 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1, which totalled 63.3%, while polyunsaturated fatty acid (PUFA) containing species totalled only 18.2%. 1-O-Alk-1'-enyl-2-acyl-GPE from cod brain was much more unsaturated with PUFA containing species totalling 52.6%, of which 18∶0a/20∶5n-3, 18∶1a/20∶5n-3 and 18∶1a/22∶6n-3 were predominant. In cod 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1 were the only other species present at over 5% each, totalling 31.8%. In both cod and trout, small amounts of species containing 22∶4n-6 were found. The results of this and earlier studies indicate that there is considerable specificity of composition at the level of molecular species between different lipid classes and subclasses.

Molecular speciation of fish sperm phospholipids: large amounts of dipolyunsaturated phosphatidylserine.

The molecular species compositions of the main diacyl phosphoglyceride classes and ether-linked subclasses from sperm of three species of fish, sea bass Dicentrarchus labrax, Atlantic salmon Salmo salar and Chinook salmon Onchorhynchus tsawytscha, were determined. The phospholipids from sperm were highly unsaturated, dipolyunsaturated fatty acid (diPUFA) molecular species comprised 64.6 to 71.8% of phosphatidylserine (PS), 10.1 to 17.4% of phosphatidylethanolamine (PE), and 3.3 to 10.1% of phosphatidylcholine (PC). In sea bass sperm, di22:6n-3 phospholipid was the predominant diPUFA molecular species, but in both salmon species 22:5n-3/22:6n-3 was also a major constituent of PS. Phospholipids containing 22:6n-3 dominated in sea bass sperm with 16:0/22:6n-3 as a major component of PC and PE, and 18:0/22:6n-3 of PE and PS in addition to di22:6n-3 in the latter two classes. In contrast, both salmon species contained much more 20:5n-3 and less 22:6n-3 so that saturated/20:5n-3 and monounsaturated/20:5n-3 molecular species were more abundant than the corresponding molecules containing 22:6n-3. Ether-linked lipids comprised 11.3-36.3% of choline and ethanolamine phosphoglycerides in each fish species. Molecular species containing 22:6n-3 were the major components of 1-O-alkyl-2-acyl-glycerophosphocholine, especially 16:0a/22:6n-3 in sea bass and 18:1a/22:6n-3 in the two salmon species, while in 1-O-alk-1'-enyl-2-acyl-glycerophosphoethanolamine, 16:0a/22:6n-3 was the major component in both salmon and 18:0a/22:6n-3 in sea bass with 18:1a/22:6n-3 abundant in all three species. In Atlantic salmon 1-O-alkyl-2-acyl-glycerophosphoethanolamine comprised 24.6% of ethanolamine glycerophospholipids which were predominantly 16:0a/22:6n-3 and 18:1a/22:6n-3. Phosphatidylinositol from sperm was dominated by stearoyl/C20 PUFA molecular species, in sea bass overwhelmingly 18:0/20:4n-6, while in both salmon species 18:0/20:4n-6 and 18:0/20:5n-3 were equally abundant.

Pyloric ceca are significant sites of newly synthesized 22:6n-3 in rainbow trout (Oncorhynchus mykiss).

In this pulse-chase study, rainbow trout fed a diet containing deuterated (D5) (17,17,18,18,18)-18:3n-3 ethyl ester accumulated D5-22:6n-3 in pyloric ceca to a greater extent than in liver 2 d post-dose. The ratio of newly synthesized D5-22:6n-3 in ceca to that in liver 2 d after feeding D5-18:3n-3 was 4.7 +/- 1.2 when expressed as per mg tissue and 5.2 +/- 2.4 when expressed as per mg protein. The amount of D5-22:6n-3 in ceca then declined whereas that in liver and blood increased, with the ratio of ceca to liver falling to 1.7 and 1.4, respectively, by day 5 and approaching unity by day 9. A crude cecal mucosa fraction contained 123 +/- 50 ng D5-22:6n-3/mg protein/mg D5-18:3n-3 eaten 2 d after feeding the tracer, compared with 35 +/- 21 ng D5-22:6n-3/mg protein/mg D5-18:3n-3 eaten in liver. Three days later the amount in cecal mucosa had fallen by one-third and that in liver had increased threefold. Most of the D5-18:3n-3 was catabolized very rapidly. The ratio of D5-18:3n-3 to 21:4n-6 (a relatively inert FA marker) in the diet was 4.0, but this fell to 0.30 in ceca and ca. 0.8 in liver, blood, and whole carcass one day after feeding. These results indicate that ceca are active in the synthesis of 22:6n-3 and the oxidation of 18:3n-3.

Biosynthesis and tissue deposition of docosahexaenoic acid (22:6n-3) in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) weighing ca. 5 g and previously acclimated for 8 wk on a diet comprising vegetable oil (11%), fish meal (5%), and casein (48%) as the major constituents were fed a pulse of diet containing deuterated (D5) (17,17,18,18,18)-18:3n-3 ethyl ester. The synthesis and tissue distribution of D5-22:6n-3 was determined 3, 7, 14, 24, and 35 d after the pulse. The whole-body accumulation of D5-22:6n-3 was linear over the first 7 d, corresponding to a rate of 0.54 +/- 0.12 microg D5-22:6n-3/g fish/mg D5-18:3n-3 eaten/d. Maximal accretion of D5-22:6n-3 was 4.3 +/- 1.2 microg/g fish/mg of D5-18:3n-3 eaten after 14 d. The amount of D5-22:6n-3 peaked in liver at day 7, in brain and eyes at day 24, and plateaued after day 14 in visceral and eye socket adipose tissue and in the whole fish. The majority of D5-22:6n-3 was found in the carcass (remaining tissues minus the above tissues analyzed separately) at all times. On a per milligram lipid basis, liver and eyes had the highest concentration of D5-22:6n-3. The experimental diet also contained 21:4n-6 ethyl ester as a marker to estimate the amount of food eaten by individual fish. From such estimates it was calculated that the great majority of the D5-tracer was catabolized, with the combined recovery of D5-18:3n-3 plus D5-22:6n-3 being 2.6%. The recovery of 21:4n-6 was 57.6%. The concentration of 22:6n-3 in the fish decreased during the 13-wk period, and the amount of 22:6n-3 synthesized from 18:3n-3 was only about 5% of that obtained directly from the fish meal in the diet.

Biosynthesis of eicosapentaenoic acid in the sea urchin Psammechinus miliaris.

The sea urchin Psammechinus miliaris (Gmelin) (Echinodermata: Echinoidea) was shown by using a deuterated tracer (D5-18:3n-3) and quantitation by negative chemical ionization gas chromatography-mass spectrometry to convert 18:3n-3 to 20:5n-3. The rate of conversion was very slow, corresponding to 0.09 microg/g tissue/mg 18:3n-3 eaten over 14 d. Deuterated arachidonic acid (D8-20:4n-6) was also included in the diet to give a measure of the relative amounts of diet eaten by the different animals. The recovery of this fatty acid in tissue lipids was 33.7% compared with only 0.95% recovery of D5-18:3n-3 and its anabolites, indicating that the majority of the D5-tracer was catabolized. Considerable elongation of D5-18:3n-3 into 20:3n-3 and a trace of 22:3n-3 was found, and these were accompanied by minor amounts of the intermediates 18:4n-3 and 20:4n-3. No deuterated 22:6n-3 was found.

(n-3) and (n-6) polyunsaturated fatty acids in the phosphoglycerides of salt-secreting epithelia from two marine fish species.

Fatty acid analyses were carried out on phosphoglycerides isolated from microsomal fractions of the rectal gland of the dogfish,Scyliorthinus canicula, and gills of the cod,Gadus morhua. Ratios of (n-3)/(n-6) polyunsaturated fatty acids were ca. 10 for phosphatidylcholine, (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from cod gills, reflecting high concentrations of 20∶5 (n-3) and 22∶6(n-3). The ratio for phosphatidylinositol (PI) from cod gills was 1.3, reflecting high concentrations of 20∶4(n-6) as well as (n-3) polyunsaturates. PC, PE and PS from rectal glands all had much lower (n-3)/(n-6) ratios than in cod gills, reflecting higher concentrations of 20∶4(n-6), but the lowest ratio was again present in PI. The latter phospholipid had high concentrations of 18∶0 in both tissues. The relative constancy of the fatty acid composition of PI in the two salt-secreting tissues and its similarity to mammalian phospholipids is considered to reflect its specialized role in biomembranes.

Lipid composition of the pineal organ from rainbow trout (Oncorhynchus mykiss).

The lipid composition of the pineal organ from the rainbow trout (Oncorhynchus mykiss) was determined to establish whether the involvement of this organ in the control of circadian rhythms is reflected by specific adaptations of lipid composition. Lipid comprised 4.9% of the tissue wet weight and triacylglycerols were the major lipid class present (47% of total lipid). Phosphatidylcholine (PC) was the principal polar lipid, and smaller proportions of other phospholipids and cholesterol were also present. Plasmalogens contributed 11% of the ethanolamine glycerophospholipids (EGP). No cerebrosides were detected. The fatty acid composition of triacylglycerols was generally similar to that of total lipids in which saturated, monounsaturated and polyunsaturated fatty acids (PUFA) were present in almost equal proportions. Each of the polar lipid classes had a specific fatty acid composition. With the exception of phosphatidylinositol (PI), in which 20∶4n-6 comprised 27.4% of the total fatty acids, 22∶6n-3 was the principal PUFA in all lipid classes. The proportion of 20∶5n-3 never exceeded 6.0% of the fatty acids in any lipid class. The predominant molecular species of PC were 16∶0/22∶6n-3 and 16∶0/18∶1, which accounted for 33.2 and 28.5%, respectively, of the total molecular species of this phospholipid. Phosphatidylethanolamine (PE) contained the highest level of di-22∶6n-3 (13.0%) of any phospholipid. There was also 4.9% of this molecular species in phosphatidylserine (PS) and 4.1% in PC. In PE, the species 16∶0/22∶6, 18∶1/22∶6 and 18∶0/22∶6 totalled 45.1%, while in PS 18∶0/22∶6 accounted for 43.9% of the total molecular species. The most abundant molecular species of PI was 18∶0/20∶4n-6 (37.8%). The lipid composition of the pineal organ of trout, and particularly the molecular species composition of PI, is more similar to the composition of the retina than that of the brain.

Dietary deficiency of docosahexaenoic acid impairs vision at low light intensities in juvenile herring (Clupea harengus L.).

In the retina of herring (Clupea harengus L.), rods are recruited from about 8 wk after hatching, and from this time there is a linear relationship between the number of rods in the photoreceptor cell population and the content of di22:6n-3 molecular species of phospholipids. Juvenile herring were reared from four weeks' post-hatching for 15 wk on either Artemia nauplii deficient in 22:6n-3 or on enriched Artemia nauplii containing 4.3% 22:6n-3. The visual performance of the fish was then determined at three light intensities (0.01, 0.1, and 1.0 lux) by observing their frequency of striking at live Artemia nauplii using infrared video recording. Herring reared on the diet containing no 22:6n-3 were less active predators, especially at the lowest light intensity where very few strikes were observed. The eyes of these fish contained greatly reduced levels of di22:6n-3 molecular species of total phospholipid, 2.1% vs. 12.0% in fish supplemented with 22:6n-3. The contribution of saturated and monounsaturated fatty acids in the molecular species of phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) was virtually unchanged, while 20:5n-3 and 22:5n-3 largely replaced 22:6n-3. There was an almost complete disappearance of di22:6n-3 PC, while the amounts of di22:6n-3 PE and PS fell by 18.1 and 20.6% to 2.7 and 7.6%, respectively. The dipolyunsaturated molecular species di20:5n-3, 20:5n-3/22:5n-3, and di22:5n-3 made up a substantial part of the deficit. We conclude that a dietary deficiency of 22:6n-3 during the period early in rod development impairs visual performance such that the fish can no longer feed at low light intensities.

The uptake from fresh water and subsequent clearance of a vanadium burden by the common eel (Anguilla anguilla).

The uptake of 48V vanadium from a solution of 10(-5) M 48V-orthovanadate by fresh-water elvers and the subsequent depletion of the vanadium burden was studied. At the end of the 8-week loading period, the levels of 48V were still increasing in the liver, kidney, bone and carcase. The uptake rate for the whole fish over the 8-week period was 760 pg atom/h/100 g body wt and the depletion rate over the following 5 weeks in clean water was about one tenth of this. Liver contained the highest amount of 48V at the end of the 8-week loading period, calculated as equivalent to 1.1 x 10(-4) g atom V/kg wet wt, and this level was unchanged at the end of the 5-week depletion period. Less than 1% of the carcase 48V was present in the fraction of MW under 2000.

Effects of the fatty acid composition of phosphatidylserine and diacylglycerol on the in vitro activity of protein kinase C from rat spleen: influences of (n-3) and (n-6) polyunsaturated fatty acids.

Protein kinase C from rat spleen was assayed with different phosphatidylserines (PtdSer) and diacylglycerols (DAG): PtdSer from bovine brain containing 0.8% 20:4 (n-6), 1.0% 20:5 (n-3) and 5.7% 22:6 (n-3); PtdSer from trout liver lacking 20:4 (n-6) and containing 0.6% 20:5 (n-3) and 43% 22:6 (n-3); 1-stearoyl-2-arachidonylglycerol prepared from synthetic 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine; DAG, prepared from cod roe phospholipids, containing 2.1% 20:4 (n-6), 11.7% 20:5 (n-3) and 29.0% 22:6 (n-3); 1,2-dioleoylglycerol. When assayed with Ca2+ in the absence of DAG there was no difference in the activity of protein kinase C between the two PtdSer. When assayed with Ca2+ in the absence of PtdSer the (n-6)-rich DAG was 2 fold more active, and the (n-3)-rich DAG 1.3 fold more active than 1,2-dioleoylglycerol. When assayed in the presence of both PtdSer and DAG, the enzyme was equally active with all of the DAG, but was 1.3 fold more active with the PtdSer from bovine brain than with the PtdSer from trout liver.