A global cline in a colour polymorphism suggests a limited contribution of gene flow towards the recovery of a heavily exploited marine mammal.

Evaluating how populations are connected by migration is important for understanding species resilience because gene flow can facilitate recovery from demographic declines. We therefore investigated the extent to which migration may have contributed to the global recovery of the Antarctic fur seal (Arctocephalus gazella), a circumpolar distributed marine mammal that was brought to the brink of extinction by the sealing industry in the eighteenth and nineteenth centuries. It is widely believed that animals emigrating from South Georgia, where a relict population escaped sealing, contributed to the re-establishment of formerly occupied breeding colonies across the geographical range of the species. To investigate this, we interrogated a genetic polymorphism (S291F) in the melanocortin 1 receptor gene, which is responsible for a cream-coloured phenotype that is relatively abundant at South Georgia and which appears to have recently spread to localities as far afield as Marion Island in the sub-Antarctic Indian Ocean. By sequencing a short region of this gene in 1492 pups from eight breeding colonies, we showed that S291F frequency rapidly declines with increasing geographical distance from South Georgia, consistent with locally restricted gene flow from South Georgia mainly to the South Shetland Islands and Bouvetøya. The S291F allele was not detected farther afield, suggesting that although emigrants from South Georgia may have been locally important, they are unlikely to have played a major role in the recovery of geographically more distant populations.

Demographic histories and genetic diversity across pinnipeds are shaped by human exploitation, ecology and life-history.

A central paradigm in conservation biology is that population bottlenecks reduce genetic diversity and population viability. In an era of biodiversity loss and climate change, understanding the determinants and consequences of bottlenecks is therefore an important challenge. However, as most studies focus on single species, the multitude of potential drivers and the consequences of bottlenecks remain elusive. Here, we combined genetic data from over 11,000 individuals of 30 pinniped species with demographic, ecological and life history data to evaluate the consequences of commercial exploitation by 18th and 19th century sealers. We show that around one third of these species exhibit strong signatures of recent population declines. Bottleneck strength is associated with breeding habitat and mating system variation, and together with global abundance explains much of the variation in genetic diversity across species. Overall, bottleneck intensity is unrelated to IUCN status, although the three most heavily bottlenecked species are endangered. Our study reveals an unforeseen interplay between human exploitation, animal biology, demographic declines and genetic diversity.

A draft fur seal genome provides insights into factors affecting SNP validation and how to mitigate them.

Custom genotyping arrays provide a flexible and accurate means of genotyping single nucleotide polymorphisms (SNPs) in a large number of individuals of essentially any organism. However, validation rates, defined as the proportion of putative SNPs that are verified to be polymorphic in a population, are often very low. A number of potential causes of assay failure have been identified, but none have been explored systematically. In particular, as SNPs are often developed from transcriptomes, parameters relating to the genomic context are rarely taken into account. Here, we assembled a draft Antarctic fur seal (Arctocephalus gazella) genome (assembly size: 2.41 Gb; scaffold/contig N50 : 3.1 Mb/27.5 kb). We then used this resource to map the probe sequences of 144 putative SNPs genotyped in 480 individuals. The number of probe-to-genome mappings and alignment length together explained almost a third of the variation in validation success, indicating that sequence uniqueness and proximity to intron-exon boundaries play an important role. The same pattern was found after mapping the probe sequences to the Walrus and Weddell seal genomes, suggesting that the genomes of species divergent by as much as 23 million years can hold information relevant to SNP validation outcomes. Additionally, reanalysis of genotyping data from seven previous studies found the same two variables to be significantly associated with SNP validation success across a variety of taxa. Finally, our study reveals considerable scope for validation rates to be improved, either by simply filtering for SNPs whose flanking sequences align uniquely and completely to a reference genome, or through predictive modelling.

Phosphatidate phosphatase--a key enzyme in glycerolipid biosynthesis. Studies on the yeast enzyme.

Phosphatidate phosphatase is an important enzyme in glycerolipid biosynthesis, but difficult to purify. A purified preparation of N-ethylmaleimide-sensitive phosphatidate phosphatase from the yeast Saccharomyces cerevisiae was obtained by a five-step protocol, using chromatography on DE-53/DEAE FF, Affi-Gel Blue, hydroxyapatite, Mono-Q, and Superdex 200. A protease-deficient yeast strain gave preparations similar to those of the wild-type strain. In exclusion chromatography, the enzyme activity of all preparations eluted at approximately the same position as albumin. However, the behavior on SDS/PAGE differed considerably among preparations, suggesting a multimeric subunit structure or degradation during purification. A 35-kDa and a 40-kDa protein band which coincided with activity were found in all preparations. Glycerol in the buffers could be excluded without rapid loss of enzyme activity, and Tris could be substituted for ammonium bicarbonate, while at least 0.6% sodium cholate in the buffers was essential.

Hepatic cholesterol metabolism in experimental nephrotic syndrome.

Hypercholesterolemia is a consistent feature of the nephrotic syndrome. However, the mechanisms underlying this perturbation are unclear. In the present work, we have investigated different factors that influence hepatic cholesterol metabolism using the nephrotic rat as a model. The induction of nephrosis resulted in a severe and sustained hypercholesterolemia. However, no effect on the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl CoA reductase, could be detected. Further, plasma lathosterol/cholesterol ratio, a measure of cholesterol synthesis, was not altered. Also, plasma levels of mevalonate, both a substrate for cholesterogenesis beyond the rate-limiting step and a marker for cholesterol synthesis, did not differ between control rats and those with established hypercholesterolemia. There was no detectable change in the expression of low density lipoprotein (LDL) receptor between the two experimental groups. We conclude that the early increase in cholesterol synthesis reported after the induction of nephrosis is not necessary for the maintenance of hypercholesterolemia. Established hypercholesterolemia of the nephrotic syndrome seems to represent a steady state in which neither enhanced hepatic cholesterol synthesis nor retarded LDL cholesterol clearance is of major importance.

On the interrelationship between hepatic carnitine, fatty acid oxidation, and triglyceride biosynthesis in nephrosis.

The nephrotic syndrome is associated with disturbances in plasma lipid pattern and metabolism. However, the reason for these perturbations is poorly understood. In the present study, we have investigated hepatic triglyceride metabolism in puromycin aminonucleoside-induced nephrotic syndrome in rats. Nephrotic rats displayed a 70% increase in hepatic triglyceride levels compared to controls (16.9 +/- 1.6 vs. 9.8 +/- 0.6 mumol/g liver; means +/- SEM, P < 0.01). The capacity for hepatic mitochondrial beta-oxidation of fatty acids was substantially elevated (80%). This was associated with a rise in the liver content of the fatty acid carrier carnitine (1.24 +/- 0.06 vs. 0.85 +/- 0.07 mumol/g dry weight, P < 0.05). A positive correlation between the levels of acetylcarnitine and acetyl-CoA was found in normal as well as in nephrotic rats, implying that carnitine plays an important role as an acetyl group acceptor in the liver under normo- and hyperlipidemic conditions. Changes in carnitine levels seem to be tightly coupled to the rate of fatty acid oxidation. There was a significant elevation in the activity of phosphatidate phosphohydrolase (E.C. 3.1.3.4) in liver microsomes from nephrotic rats (1.07 +/- 0.09 vs. 0.81 +/- 0.04 nmol/min.mg protein, P < 0.02). Hepatic very low density lipoprotein (VLDL)-triglyceride secretion rate was 18% higher in nephrotic rats than in controls. The results demonstrate a deranged hepatic triglyceride metabolism in nephrosis, with an increased hepatic triglyceride biosynthesis, a sizable accumulation of hepatic triglycerides, and only a modest increase in VLDL triglyceride secretion. In addition, mitochondrial beta-oxidation of fatty acids was enhanced, associated with an increased availability of carnitine.

Difficulties in the assay of phosphatidate phosphohydrolase activity. Influence of ionic strength, detergent, and selection of substrate.

In the present paper, problems in connection with assay of the activity of magnesium-dependent rat liver phosphatidate phosphohydrolase (PAP) are discussed. PAP activity is usually measured by following the production of diacylglycerol or inorganic phosphate from the substrate phosphatidate. These two methods may give widely different results due to a number of factors that may affect the assay. One such factor is the composition of the substrate. Higher apparent enzyme activity was observed with dioleoyl-phosphatidate than with dipalmitoyl-phosphatidate. This substrate-dependent difference in apparent PAP activity was 2-2.5-fold in the absence and 10-fold in the presence of Triton X-100, respectively. Triton X-100 reduced the activity as measured with the dipalmitoyl-phosphatidate substrate. In contrast, the activity of PAP as measured with dioleoyl-phosphatidate was stimulated by Triton X-100. The stimulatory effect of Triton was reduced or abolished when the ionic strength in the assay mixture was increased. Assays based on 32P-labeled substrate are rapid and sensitive. It is shown here that 33P can be used as an alternative. This radionuclide has a longer half-life and also emits particles with lower energy, thus posing less potential health hazards for the user.

Studies on rat liver phosphatidate phosphohydrolase and plasma lipids: effect of HMG-CoA reductase inhibitors.

Normolipidemic rats were treated with HMG-CoA reductase inhibitors (lovastatin or pravastatin) for periods of 1-3 days. Administration of these drugs reduced plasma triacylglycerol levels. Lovastatin-treated rats displayed a 30% lower hepatic triacylglycerol secretion rate compared to controls as estimated using Triton WR-1339. Lovastatin in a dose of 0.1% in the diet for 3 days increased hepatic phosphatidate phosphohydrolase (PAP) activity 2- to 3-fold, both in the cytosolic and microsomal fractions. Similar effects were seen in the presence of 0.2% pravastatin. PAP in both fractions was stimulated to a lesser extent by treatment with 0.1% pravastatin. These differences in PAP activity obtained by different treatment regimens were preserved during purification of cytosolic PAP on hydroxylapatite. The increase in PAP activity upon treatment with lovastatin or pravastatin was gradual and occurred simultaneously with the apparent increase in HMG-CoA reductase activity. In rats treated with the reductase inhibitors, the activity of microsomal and cytosolic PAP was inversely correlated to plasma triacylglycerol levels. The results indicate that hepatic triacylglycerol and cholesterol synthesis might be co-ordinately regulated, and also suggest that the activity of PAP is rapidly modulated in concert with changes in plasma triacylglycerol levels.

Studies on triglyceride metabolism: phosphatidate phosphohydrolase from guinea pig harderian gland.

The guinea pig Harderian gland, located in the orbit, is characterized by a high production of lipids. However, little is known about the regulation of the metabolic pathways involved. In the present paper the properties of guinea pig Harderian gland phosphatidate phosphohydrolase, a key enzyme in triglyceride biosynthesis, was investigated. The enzyme was present both in the cytosolic and the microsomal fraction from the gland. Cytosolic phosphatidate phosphohydrolase was purified by hydroxylapatite chromatography. The enzyme was dependent on magnesium ions and inhibited in the presence of fluoride. The dependence on the substrate phosphatidate was investigated and the apparent Km for phosphatidate was about 0.6 mM. Phosphatidate phosphohydrolase activity was influenced by different phospholipids. As is the case for the rat liver enzyme, phosphatidylethanolamine was found to stimulate the enzyme activity. The results indicate that Harderian gland phosphatidate phosphohydrolase has similar properties as the corresponding enzyme from rat liver, suggesting that it may be of regulatory importance.

Stimulation and inhibition of the activity of rat liver cytosolic phosphatidate phosphohydrolase by various phospholipids.

The influence of phospholipids on the activity of the soluble phosphatidate phosphohydrolase from rat liver was studied. Phosphatidylethanolamine stimulated the enzyme activity whereas phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol were inhibitory. At a phospholipid concentration of 0.7 mg/ml, phosphatidylglycerol inhibited phosphatidate phosphohydrolase activity by 75%, while the enzyme activity was stimulated twofold in the presence of phosphatidylethanolamine. Both lysophosphatidylglycerol and lysophosphatidylethanolamine inhibited phosphatidate phosphohydrolase activity as did octylglucoside, sodium cholate, and Tween 20. The finding that phospholipids influence hepatic phosphatidate phosphohydrolase activity indicates that changes in the lipid environment may modulate the enzyme activity.

Effect of phosphorylation in vitro of human fibrinogen with protein kinase C on thrombin-induced gelation.

Thrombin-induced gel formation of fibrinogen phosphorylated by protein kinase C yielded a transparent gel, whereas unphosphorylated fibrinogen yielded a coarse gel. The mass-length ratio was found to be one order of magnitude higher for the unphosphorylated than for the phosphorylated fibrinogen. Since the phosphorylated sites are located near the cross-linking sites in the A alpha-chain of fibrinogen, it is likely that the introduction of charged phosphate groups in this region prevent the lateral growth of the fibrin fibres.

Phosphorylation of human fibrinogen in vitro with protein kinase C: characterization of the phosphorylated sites.

Phosphorylation of human fibrinogen in vitro by incubation with [gamma-32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [32P]phosphate at serine residues located in the A alpha-chain. In order to identify the residues that were phosphorylated, the A alpha-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser557, Ser558, Ser559, and Ser599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the A alpha-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln328 and Gln366, in this portion of the A alpha-chain take part in the cross-linking of fibrin.

Aspects on the phosphorylation of muscle phosphofructokinase by protein kinase C--inhibition by phosphofructokinase stabilisers.

Our report presents data on the phosphorylation of muscle phosphofructokinase by Ca2+-activated, phospholipid-dependent protein kinase. We have found a stoichiometrical phosphorylation (about 1.5 mol per mol subunit), and a low apparent Km (about 0.7 microM). These data speak in favor of a physiological role for the reaction, as does the fact that phosphofructokinase from a new species (rat) was successfully phosphorylated. On the other hand we present the hitherto unpublished circumstance that the phosphorylation is inhibited by conditions that stabilise the activity of phosphofructokinase. This fact makes us question the true significance of this reaction.

Phosphorylation in vitro of fibrinogen from three mammalian species with four different protein kinases.

Human, dog, and rabbit fibrinogen served as substrates for calcium-activated, phospholipid-dependent protein kinase, cAMP-dependent protein kinase, casein kinase TS, and casein kinase S. The chains of phosphorylated fibrinogen were separated by polyacrylamide gel electrophoresis and the phosphorylation patterns, obtained on autoradiography of the gels, were found to be characteristic for each of the four protein kinases. Dog, and even more so rabbit, fibrinogen was phosphorylated more rapidly than human fibrinogen by calcium-activated, phospholipid-dependent protein kinase and by casein kinase TS. Dog fibrinogen was not a good substrate for cAMP-dependent protein kinase. The rate of phosphorylation with casein kinase S did not differ very much between the fibrinogens of the three species. In most cases the A alpha-chain was most rapidly phosphorylated. However, in dog fibrinogen incubated with casein kinase TS the B beta-chain was most rapidly phosphorylated. A substantial part of this phosphate seemed to be incorporated as phosphorylthreonine into fibrinopeptide B. In human fibrinogen incubated with the casein kinase TS preparation the gamma-chain as well as the A alpha-chain appeared to be phosphorylated.

Phosphorylation of human fibrinogen in vitro by calcium-activated phospholipid-dependent protein kinase from pig spleen.

Human plasma fibrinogen rapidly incorporated stoichiometric amounts of [32P]-phosphate when incubated with [32P]ATP and calcium-activated, phospholipid-dependent protein kinase purified from pig spleen. Half-maximal fibrinogen kinase activity was attained at less than 0.1 mM calcium acetate. The optimum concentration of phosphatidylserine was about 50 micrograms per ml. Diolein slightly potentiated the stimulatory effect of phosphatidylserine. The alpha-chain of fibrinogen, which is reported to contain endogenous phosphate (Blombäck, B., Blombäck, M., Edman, P., & Hessel, B. (1962) Nature 193, 883-884 and Doolittle, R.F., Watt, K.W.K., Cottrell, B.A., Strong, D.D., & Riley, M. (1979) Nature 280, 464-468) was phosphorylated by the protein kinase. The apparent Km value for the phosphorylation reaction (0.3-0.6 microM fibrinogen) was comparable with the Km values reported for the hitherto most effective substrate proteins for protein kinase C. Up to 5 mol phosphate per mol fibrinogen could be incorporated, indicating at least three phosphorylatable sites per half molecule.

Phosphopeptide substrates of a phosphoprotein phosphatase from rat liver.

The substrate specificity of a preparation of phosphoprotein phosphatase (Mr = 32 000) from rat liver was investigated. Phosphopeptides based on the structure Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Glx-Leu and Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-Val-Tyr-Glu-Pro-Leu-Lys were used. These phosphopeptides correspond to the phosphorylation sites of rat liver pyruvate kinase (type L) and the beta subunit of rabbit muscle phosphorylase b kinase, respectively. A decrease in the apparent Km values and a concomitant increase in Vmax values was observed when the number of amino acyl residues after the phosphoseryl residue in the respective phosphopeptides were increased from 2 to 4, 5, or 6. Most of the phosphopeptides investigated generally showed apparent Km values higher than the values obtained with phosphopyruvate kinase. Ala-Ser(P)-Val-Ala and Gly-Ser(P)-Val-Tyr appeared to be the shortest phosphopeptides that could be dephosphorylated rapidly. These findings support the hypothesis that a small part of the phosphoprotein may be sufficient to fulfill the minimal requirements for its dephosphorylation.

Amino acid sequence around the phosphorylated sites of porcine and rat liver pyruvate kinase, type L.

Chymotrypsin removed the phosphorylated site of porcine liver pyruvate kinase without inactivating the enzyme. The amino acid sequence of the phosphopeptide obtained was analyzed. By analysis of CNBr fragments containing 33 amino acid residues, further information was obtained on the amino acid sequence around the phosphorylated sites of porcine and rat liver pyruvate kinase. It was found to be (formula see text) for the porcine isozyme and was very similar for the rat isozyme, although the order of the five most C-terminal amino acid residues (Leu, Pro, Ala2, Homoserine) in this fragment was not resolved and Leu was exchanged for Val in position 12 and Arg for Gln in position 26. The chymotryptic porcine isozyme phosphopeptide, composed of 18 amino acid residues, was entirely contained in the corresponding CNBr fragment (residues 7-24).