Characterization of proteins and fatty acid composition in Galapagos fur seal milk. Occurrence of whey and casein protein polymorphisms

1. Milk proteins of the Galapagos fur seal (Arctocephalus galapagoensis) were separated adequately into whey and casein fractions using bovine milk analysis methods. 2. In samples from days 5-30 of lactation 40% of the total proteins were whey and 60% caseins; in mid-lactation, day 150, 25% were whey and 75% casein proteins. 3. Electrophoretic and isoelectric focusing patterns of fur seal whey protein differed widely from bovine patterns, whereas those of caseins were similar. 4. Polymorphisms of fur seal whey and casein proteins were noted and did not seem related to different stages of lactation. 5. C-16 and C-18 fatty acids contributed about 70% of fatty acids; 63% of the total acids in milk fat were unsaturated.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.     

Fatty acid content of depot fat in the northern fur seal (Callorhinus ursinus)

The fatty acid content of depot fat samples from 15 northern fur seals (Callorhinus ursinus) were determined by gas-liquid chromatography. Callorhinus ursinus has a high proportion of short chain saturated acids: C10, C11, C12, C13, C15. Unsaturated longer chain acids C16:1, and C16:2, and C18:1 also were found. Results obtained are compared to a previously reported milk lipid analysis of the northern fur seal.     

The impact of dietary fats,photoperiod, temperature and season on morphological variables,torpor patterns,and brown adipose tissue fatty acid composition of hamsters,Phodopus sungorus

We investigated how dietary fats and oils of different fatty acid composition influence the seasonal change of body mass, fur colour, testes size and torpor in Djungarian hamsters, Phodopus sungorus, maintained from autumn to winter under different photoperiods and temperature regimes. Dietary fatty acids influenced the occurrence of spontaneous torpor (food and water ad libitum) in P. sungorus maintained at 18°C under natural and artificial short photoperiods. Torpor was most pronounced in individuals on a diet containing 10% safflower oil (rich in polyunsaturated fatty acids), intermediate in individuals on a diet containing 10% olive oil (rich in monounsaturated fatty acids) and least pronounced in individuals on a diet containing 10% coconut fat (rich in saturated fatty acids). Torpor in P. sungorus on chow containing no added fat or oil was intermediate between those on coconut fat and olive oil. Dietary fatty acids had little effect on torpor in animals maintained at 23°C. Body mass, fur colour and testes size were also little affected by dietary fatty acids. The fatty acid composition of brown fat from hamsters maintained at 18°C and under natural photoperiod strongly reflected that of the dietary fatty acids. Our study suggests that the seasonal change of body mass, fur colour and testes size are not significantly affected by dietary fatty acids. However, dietary fats influence the occurrence of torpor in individuals maintained at low temperatures and that have been photoperiodically primed for the display of torpor.Abbreviations BAT brown adipose tissue - bm body mass - FA fatty acid(s) - MR metabolic rate - MUFA monounsaturated fatty acid(s) - PUFA polyunsaturated fatty acid(s) - SFA saturated fatty acid(s) - T _a air temperature - T _b body temperature - T_s body surface temperature(s) - TNZ thermoneutral zone - UFA unsaturated fatty acid(s)     

A platypus' eye view of the mammalian genome

The genome of monotremes, like the animals themselves, is unique and strange. The importance of monotremes to genomics depends on their position as the earliest offshoot of the mammalian lineage. Although there has been controversy in the literature over the phylogenetic position of monotremes, this traditional interpretation is now confirmed by recent sequence comparisons. Characterizing the monotreme genome will therefore be important for studying the evolution and organization of the mammalian genome, and the proposal to sequence the platypus genome has been received enthusiastically by the genomics community. Recent investigations of X-chromosome inactivation, genomic imprinting and sex chromosome evolution provide good examples of the power of the monotreme genome to inform us about mammalian genome organization and evolution.     

The androgen receptor gene is located on a highly conserved region of the X chromosomes of marsupial and monotreme as well as eutherian mammals

The androgen receptor gene (AR), which is located on the long arm of the human X chromosome, was mapped by somatic cell analysis and in situ hybridization in marsupial and monotreme species. Both methods demonstrated that it was located on the X chromosome in each marsupial species, and also in the platypus. We conclude that this gene is part of a highly conserved region of the mammalian X, represented by the human Xq, which formed part of the X chromosome in a mammalian ancestor 150 million years ago. Since this gene is located proximally on the long arm of the monotreme X, which is G-band homologous to the Y and apparently exempt from X chromosome inactivation, the conservation of this region has evidently not depended on its isolation by X-Y differentiation or on X inactivation.     

Molecular characterization of cathepsin L from hepatopancreas of the carp Cyprinus carpio

Purified cathepsin L from carp, Cyprinus carpio, consists of a 28 kDa single-chain form that is different from the 24 and 5 kDa mammalian two-chain form. We cloned cathepsin L from carp hepatopancreas. The sequence consisted of a 1490 bp cDNA and a 1014 bp open reading frame, encoding a deduced protein of 337 amino acids that is likely processed to an active enzyme (single-chain form) with 222 amino acids. Its similarity to other types of vertebrate cathepsin L is less than 69%. Mammalian cathepsin L is further processed to a two-chain form, but possibly this is not the case with carp cathepsin L: the P1 site where cleavage occurred in the two-chain form of mammalian cathepsin L contains a serine, while carp cathepsin L processes a valine. Therefore, carp cathepsin L may have a different mechanism of action from mammalian cathepsin L.     

Fatty acid metabolism of isolated mammalian cells

It is now clear that a wide variety of differentiated cells in culture exhibit essentially the full spectrum of mammalian fatty acid metabolism. These cells readily incorporate free fatty acids into membrane phosphoglycerides, modify exogenous fatty acids by desaturation and elongation, and store excess fatty acyl groups, primarily as triacylglycerols. Similarly, many different types of cells synthesize cyclooxygenase and lipoxygenase derivatives of long chain polyunsaturated fatty acids. Furthermore, although the fatty acid composition of cellular phospholipids can be modified by medium supplementation, cells in culture exhibit definite fatty acyl specificities for the various steps of fatty acid activation, transesterification and release. As the extensive repertoire of fatty acid metabolism in mammalian cells has been elucidated, and as the ability to grow differentiated cells in culture has increased, new questions have arisen. There is still much to be learned about the enzymes involved in synthesizing and maintaining the unique fatty acid composition of the different cellular phospholipids and the processes which regulate the desaturation, elongation and retroconversion of polyunsaturated fatty acids. Other areas of great current interest are the mechanisms by which certain long chain polyunsaturated fatty acids are made available for conversion to oxygenated, biologically-active derivatives, the metabolic interactions between different polyunsaturated fatty acids, particularly n-3 and n-6 fatty acids, the cellular roles of the C22 polyunsaturated fatty acids, and the functions of particular molecular species of phospholipids in membrane-mediated events. Further research in these areas will contribute to unravelling the role of fatty acids and fatty acid derivatives in the physiological processes of mammalian cells.     

Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm

Long-chain fatty acids can transfer passively across mammalian cell membranes. However, under physiological conditions of low fatty acid to albumin ratios in the circulation, the major fraction of uptake appears to be mediated by a saturable, protein-facilitated component. A simple diffusion process becomes significant at high molar ratios of fatty acid to albumin as the concentration of free fatty acid in solution is increased. Identification of the mammalian membrane fatty acid transporter(s) has been the focus of active investigation by several research groups. In this review we discuss three candidate proteins: FABPm, FAT/CD36 and FATP which have been cloned and are currently being characterized. Recent evidence arguing for an important role of the fatty acid transport step in general metabolism and linking these proteins to physiologic or metabolic abnormalities is described.     

Development of oxaalkyne and alkyne fatty acids as novel tracers to study fatty acid beta-oxidation pathways and intermediates

Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes. Combining these new tracer tools with highly sensitive chromatography and mass spectrometry analyses, this study confirms differences in metabolic handling of fatty acids of different chain length. Unlike longer chains, we found that medium-chain fatty acids that were activated inside or outside of mitochondria by different acyl-CoA synthetases could enter mitochondria in the form of free fatty acids or as carnitine esters. Upon mitochondrial beta-oxidation, shortened acyl-carnitine metabolites were then produced and released from mitochondria. In addition, we show that hepatocytes ultimately also secreted these shortened acyl chains into their surroundings. Furthermore, when mitochondrial beta-oxidation was hindered, we show that peroxisomal beta-oxidation likely acts as a salvage pathway, thereby maintaining the levels of shortened fatty acid secretion. Taken together, we conclude that this new method based on oxaalkyne and alkyne fatty acids allows for metabolic tracing of the beta-oxidation pathway in tissue lysate and in living cells with unique coverage of metabolic intermediates and at unprecedented detail.     

Monotreme ossification sequences and the riddle of mammalian skeletal development

The developmental differences between marsupials, placentals, and monotremes are thought to be reflected in differing patterns of postcranial development and diversity. However, developmental polarities remain obscured by the rarity of monotreme data. Here, I present the first postcranial ossification sequences of the monotreme echidna and platypus, and compare these with published data from other mammals and amniotes. Strikingly, monotreme stylopodia (humerus, femur) ossify after the more distal zeugopodia (radius/ulna, tibia/fibula), resembling only the European mole among all amniotes assessed. European moles also share extreme humeral adaptations to rotation digging and/or swimming with monotremes, suggesting a causal relationship between adaptation and ossification heterochrony. Late femoral ossification with respect to tibia/fibula in monotremes and moles points toward developmental integration of the serially homologous fore- and hindlimb bones. Monotreme cervical ribs and coracoids ossify later than in most amniotes but are similarly timed as homologous ossifications in therians, where they are lost as independent bones. This loss may have been facilitated by a developmental delay of coracoids and cervical ribs at the base of mammals. The monotreme sequence, although highly derived, resembles placentals more than marsupials. Thus, marsupial postcranial development, and potentially related diversity constraints, may not represent the ancestral mammalian condition.     

Molecular evolution of monotreme and marsupial whey acidic protein genes

Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.     

FATTY ACID COMPOSITION OF FATS FROM THE URUGUAYAN FUR SEAL (ARCTOCEPHALUS AUSTRALIS ZIMMERMANN)

The composition and properties of commercial oil collected from Uruguayan fur seals ( Arctocephalus australis Zimmermann) during 1985, 1986 and 1987 are reported. Oil recovered from some organs (kidney, heart, liver) and blubber were also analyzed. All have compositions similar to oils from other marine animals in commercial use, containing high percentages of long chain and polyunsaturated fatty acids.     

A Tachyglossid-Like Humerus from the Early Cretaceous of South-Eastern Australia

A partial right humerus has been recovered from the Early Cretaceous (Albian) Eumeralla Formation at Dinosaur Cove in south-eastern Australia. General morphology, size and the presence of a single epicondylar foramen (the entepicondylar) suggest that the bone is from a mammal or an advanced therapsid reptile. The humerus is similar in size, shape and torsion to the equivalent bone of extant and late Neogene echidnas (Tachyglossidae) but, contrary to the situation in extant monotremes, in which the ulna and radius articulate with a single, largely bulbous condyle, it bears a shallow, pulley-shaped (i.e. trochlear-form) ulnar articulation that is confluent ventro-laterally with the bulbous radial condyle. This form of ulnar articulation distinguishes this bone from the humeri of most advanced therapsids and members of several major groups of Mesozoic mammals, which have a condylar ulnar articulation, but parallels the situation found in therian mammals and in some other lineages of Mesozoic mammals. As in extant monotremes the distal humerus is greatly expanded transversely and humeral torsion is strong. Transverse expansion of the distal humerus is evident in the humeri of the fossorial docodont Haldanodon, highly-fossorial talpids and some clearly fossorial dicynodont therapsids, but the fossil shows greatest overall similarity to extant monotremes and it is possible that the peculiar elbow joint of extant monotremes evolved from a condition approximating that of the fossil. On the basis of comparisons with Mesozoic and Cainozoic mammalian taxa in which humeral morphology is known, the Dinosaur Cove humerus is tentatively attributed to a monotreme. However, several apparently primitive features of the bone exclude the animal concerned from the extant families Tachyglossidae and Ornithorhynchidae and suggest that, if it is a monotreme, it is a stem-group monotreme. Whatever, the animal's true affinity, the gross morphology of its humerus indicates considerable capacity for rotation-thrust digging.     

Fatty acid interactions with proteins: what X-ray crystal and NMR solution structures tell us

The interactions of fatty acids with proteins have been studied by a variety of conventional approaches for decades. However, only limited aspects of fatty acid-protein interactions have been elucidated, even with the integration of information gleaned from the many techniques. Judgments must be made about what information is most reliable, particularly when derivatives of fatty acids are substituted for natural fatty acids. In recent years, the application of techniques of structural biology has brought about dramatic advances in this important area of lipid research. High-resolution crystallographic and NMR structures of several proteins with bound fatty acids reveal the complete tertiary structure of the protein and molecular details of fatty acid-protein interactions. The examples presented include most of the known structures of (non-enzymatic) proteins that bind fatty acids. The proteins are found in very different compartments of cells and organisms: the plasma compartment (human serum albumin); the cytosolic compartment of mammalian cells (fatty acid- binding proteins); the cytosol of plant cells (nonspecific lipid-transfer protein); the nucleus of mammalian cells (peroxisome proliferator-activated receptor and hepatic nuclear factor 4); and a bacterial membrane (halorhodopsin). This review discusses the structural features of these proteins and their binding pocket(s) and compares the specific modes of their interactions with fatty acids.     

The manipulation of fatty acid composition in L-M cell monolayers supplemented with cyclopentenyl fatty acids

Mouse L-M fibroblasts, grown in a serum-free medium, were supplemented with fatty acids of 16 and 18 carbon chain lengths that contain a cyclopentene ring in the ω position. These fatty acids, unnatural to mammalian systems, were incorporated into the major lipid classes of L-M fibroblasts. Supplementation with the cyclopentenyl fatty acids caused an accumulation of neutral glycerolipids and marked inhibition of cell growth. Following the addition of supplement, the cells became more rounded. Of particular interest was the fact that the phospholipid fraction isolated from treated cells contained cyclic fatty acids that accounted for as much as 24% of the total phospholipid acyl groups. Unlike the pattern of distribution displayed by endogenous natural monoenes, the majority of the cyclic acid present was esterified in the sn-1 position of both phosphatidylcholine and phosphatidylethanolamine. The 18-carbon cyclic fatty acid [chaulmoogric acid, 13-(2-cyclopenten-1-yl)tridecanoic acid] was incorporated at the expense of the endogenous C-16:0, C-18:0, and C-18:1 fatty acids of the glycerophospholipids. The esterification altered the ratio of saturated to unsaturated acyl groups in the cellular phospholipids. No biochemical modification of chaulmoogric acid was detected.Our results imply that incorporation of unnatural fatty acid analogs, such as chaulmoogric acid, into cellular membranes would alter the functional properties of biological membranes that are dependent on membrane fluidity and structural organization.     

Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella)

Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans. In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells.     

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the rate of sterol synthesis diminish in cultures with high cell density

The specific activity of HMG-CoA reductase, the major rate-limiting enzyme in the sterol biosynthetic pathway, declined linearly with increasing cell density in four different lines of mammalian cell cultures. As expected, this caused the rates of sterol synthesis from [14C]acetate to decline in a parallel manner. The decrease in reductase activity in the dense cultures was also correlated with decreased incorporation of [14C]acetate into fatty acids and [3H]thymidine into DNA. In contrast, the activities of two enzymes, NADH dehydrogenase and 5'-nucleotidase, which are not involved in lipid synthesis, were independent of changes in cell density. The simplest explanation for these data is tht HMG-CoA reductase and the synthesis of sterol and fatty acids are regulated in concordance with the rate of cell growth and proliferation.     

Lipids of chicken epidermis

The lipids from chicken epidermis were analyzed by a combination of quantitative thin-layer and gas-liquid chromatography and by chemical and spectroscopic methods. The lipid groups present included wax diesters (34%), triglycerides (32%), sterols (11%), phospholipids (11%), nonphosphorus-containing sphingolipids (3%), beta-D-glucosylsterols (3%), 6-O-acyl-beta-D-glucosylsterols (2%), steryl esters (1%), cholesteryl sulfate (1%), and free fatty acids (1%). The major phospholipids were phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, and the sphingolipids included ceramides, glucosylceramides, O-acylceramides, and O-acylglucosylceramides. Glucosylsterols and acylglucosylsterols have not been found in mammalian skin, and may be relevant to the evolutionary history of the epidermal water barrier. The wax diesters contained mainly 16-, 18-, and 20-carbon saturated fatty acids esterified to 20- through 24-carbon threo and erythro 2,3-diols, while the chicken epidermal triglycerides contained some very long-chain (26-40 carbon) saturated fatty acids. These wax diesters and unusual triglycerides may be of significance in human health.