Identification, isolation and characterization of epidermal lipids containing linoleic acid

Three specific linoleate-rich lipids have been identified in pig epidermis and are referred to as O-acylglucosyl ceramide, O-acyl ceramide, and O-acyl acid. The acid moiety is up to 70% linoleic acid and linked via the hydroxyl group of a omega-OH long-chain fatty acid, which itself is linked to sphingosine or glucosyl sphingosine. The identification of O-acyl ceramide confirmed the findings of another group, whereas the structural configuration of the O-acylglucosyl ceramide is different to previous reports. The identification of an O-acyl acid in epidermis is novel. Our evidence allows us to speculate that a hydroxylated derivative of the O-acyl ceramide may be intimately involved in the permeability barrier of skin, perhaps providing sufficient polarity to maintain a lamellar phase in the intercellular space of the lower stratum corneum region, and that it is specifically in this form that linoleic acid is involved in skin barrier function. Alternatively, or concurrently, this species may act as a signal for late keratinisation events.     

Calcium-regulated photoproteins of marine coelenterates

Ca(2+)-regulated photoproteins are bioluminescent proteins responsible for bioluminescence of marine coelenterates. The photoprotein molecule is a stable enzyme-substrate complex consisting of a single polypeptide chain and an oxygen "pre-activated" substrate, 2-hydroperoxycoelenterazine, which is tightly but non-covalently bound with a protein. The bioluminescence is triggered by calcium ions and originates from an oxidative decarboxylation of a protein bound substrate. The review provides current data on the photoproteins structure, the mechanism of bioluminescent reaction, the function of some amino acid residues of an active site in the catalysis and the formation of the emitter, as well as on applications of these proteins in a bioluminescent analysis.     

Calcium-regulated photoproteins of marine coelenterates

Calcium-regulated photoproteins are bioluminescent proteins that are responsible for the luminescence of marine coelenterates. A photoprotein molecule is a stable enzyme-substrate complex consisting of a single polypeptide chain and an oxygen-preactivated substrate, 2-hydroperoxycoelenterazine, which is tightly but noncovalently bound with the protein. Bioluminescence is triggered by Ca²⁺ and results from decarboxylation of the substrate bound with the protein. This review considers the current information about the structure of photoproteins, the mechanism of the bioluminescent reaction, the function of particular amino acid residues of the active center in catalysis and the formation of the emitter, and the use of photoproteins in bioluminescent microanalysis.     

Identification, isolation and characterization of canine minisatellite sequences

A Charomid ordered-array library containing a 2–16 Kb size fraction of MbeoI-digested canine genomic DNA has been screened with the Jeffreys multilocus probes, 33-6 and 33-15, to identify and isolate canine minisatellite sequences. Of the 48 positive clones identified, 7 were found to contain polymorphic mini-satellites with heterozygosities in the range 20–88%. The majority of the remainder were either monomorphic or dimorphic in the animals tested. Analysis of intrabreed variation in Bedlington Terriers using two polymorphic minisatellites has shown that a significant reduction occurs in the number of alleles seen compared to an agglomerated population sample, correlating with the high level of inbreeding within this breed. Flanking DNA sequence and partial repeat sequence is presented for the most polymorphic minisatellite thus far identified, cCfaMP5. The variable region in this mini-satellite is similar to human minisatellites which show a distinct purine or pyrimidine strand bias.     

Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization.

Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation.     

Phospholipid fatty acid composition of gorgonians of the genus Pseudopterogorgia: Identification of tetracosapolyenoic acids

The phospholipid fatty acid composition of the Caribbean gorgonians Pseudopterogorgia acerosa (Pallas), Pseudopterogorgia americana (Gmelin), Pseudopterogorgia bipinnata (Verrill) and Pseudopterogorgia rigida (Bielschowsky) is described for the first time. The main phospholipids identified were phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine. All four gorgonians presented a similar phospholipid fatty acid composition. The main fatty acids were 16:0, 18:3(n-6), 18:4(n-3), 20:4(n-6), 22:6(n-3), 24:5(n-6) and 24:6(n-3). In all of the studied Pseudopterogorgia gorgonians, high amounts of the tetracosapolyenoic fatty acids 24:5(n-6) and 24:6(n-3) were identified. In the four gorgonians studied, n-6 polyunsaturated fatty acids predominated. These results suggest that the occurrence of tetracosapolyenoic fatty acids in the Gorgoniidae is more general than previously recognized.     

The component fatty acids of some marine algal lipids

Twelve marine algae of the Rhodophyta, 17 of the Phaeophyta and 5 of the Chlorophyta were analysed for their fatty acid content. It is possible that the fatty acid distribution may be useful for taxonomic purposes. It is found, that, as in higher plants, there are specific fatty acid distributions in the galactosyl diglycerides, e.g. the ω3 acids with the highest unsaturation are concentrated in the monogalactosyl diglycerides.     

Palytoxin isolated from marine coelenterates. The inhibitory action on (Na,K)-ATPase

Palytoxin (PTX), C129H223N3O54, a highly toxic substance isolated from zoanthids of Palythoa tuberculosa, inhibited (Na,K)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) prepared from guinea pig heart and hog cerebral cortex in a dose-dependent manner at concentrations greater than 10(-8) M. In the presence of Na (100 mM) and K (20 mM), PTX showed potency nearly equal to that of ouabain. When the ATPase was activated by the various Na concentrations at a constant K concentration, both PTX and ouabain inhibited the ATPase activity noncompetitively. On the other hand, when K concentration was changed at a constant Na concentration, PTX caused a competitive inhibition in all ranges of K concentrations employed, whereas ouabain caused a competitive inhibition at low concentrations and a noncompetitive inhibition at high concentrations.     

Neutral lipids, their fatty acids, and the sterols of the marine ciliated protozoon, Parauronema acutum

The neutral lipids and their fatty acids and the sterol fractions of the marine ciliated protozoon, Parauronema acutum, were characterized. The neutral lipids consisted of triglycerides (30%), sterols (29%), free fatty acids (24%), steryl esters (9%), and diglycerides (8%) and small amounts of fatty alcohols. The fatty acid profiles of these lipids were very similar although quantitative differences were detected. Saturated fatty acids, primarily 14:0, 16:0, and 18:0 constituted 20-30% of the total. Unsaturated fatty acids containing one to three double bonds, primarily 18:1(9), 18:2 (9,12), 18:3 (9, 12, 15) and 20:3 (11, 14, 17), constituted 35-50% of the total. Highly unsaturated fatty acids, 18:4 (6, 9, 12, 15), 20:5 (5, 8, 11, 14, 17) and 22:6 (4, 7, 10, 16, 19), constituted 16-25% of the total. The fatty alcohols consisted of 14:0 (2%), 16:0 (66%), 18:0 (3%), 20:0 (8%), and 22:0 (21%). The sterols of Parauronema acutum consisted of cholesterol (53%), campesterol (32%), desmosterol (7%), and beta-sitosterol (8%).     

Bone lipids of marine fishes.

Assessment of candidates for investigation of bone lipid metabolism yielded the following findings. (1) A tropical marine butterflyfish, Chaetodon ornatissimus, had oil-filled bones (66-80% lipid, percent dry weight) hence may be a suitable condidate. (2) The tropical marine fishes Exallias brevis, Pomacentrus jenkensi, and Chromus agilis, and a Canadian fish Sebastes ruberrimus, had intermediate quantities of oil in their bones (12-49% lipid). (3) In all the foregoing species the major bone lipid was triglyceride, usually more abundant in skull than spine. Sterol and phospholipid were also present. (4) The major fatty acids of the triglycerides (and phospholipids) were 16:0, 18:0, 18:1, and C20, C22 acids. Those acids were dominated by 20:4, 20:5, 22:5, and 22:6. (5) There was more total unsaturation in the bone lipids of S. ruberrimus (from 10 degrees C water; 67-72% unsaturation) compared to the tropical fish (from 25 degrees C water; 32-67% unsaturation) with the exception of E. brevis. (6) One of the tropical species (Arothron meleagris) and a Canadian Chimaeran (Hydrolagus colliei) contained only 1-3% lipid in their bones.     

Dicyclohexylammonium salts for the isolation and characterization of aldonic acids

Dicyclohexylammonium salts of aldonic acids may be prepared from aldonolactones, as well as from metal aldonates and the free acids. Although accompanied by decomposition, their melting points are usually sharp, and these salts appear to have some potential utility for the isolation and characterization of aldonic acids.Dicyclohexylammonium 2-acetamido-2-deoxy-d-gluconate (1) has recently been described; in the course of the present investigation, it was converted into 2-acetamido-2-deoxy-d-glucose (3), confirming the configuration previously assigned to it. With aqueous dicyclohexylamine, 2-acetamido-2-deoxy-d-mannono-1,4-lactone (2) gives 1. The configuration of 2 was reconfirmed through reduction to 2-acetamido-2-deoxy-d-mannitol (4), and the optical rotations of this compound and its d-gluco isomer in acidified ammonium molybdate solution were found to be useful physical constants for distinguishing these alditols.2-Acetamido-2-deoxy-d-galactono-1,4-lactone affords a crystalline dicyclohexylammonium salt of the corresponding acid, from which the lactone may be regenerated.     

Positional distribution of fatty acids in lipids of the marine diatom Phaeodactylum tricornutum

The composition of fatty acids and lipids in the marine diatom, Phaeodactylum tricornutum was determined. The Lipids consisted of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphtidylinositol, triacylglycerol and minor unidentified ones. At the early stationary phase of growth, the total fatty acids were mainly 20:5, 16:1, 16:0 and 16:3. 20:5 was distributed in polar lipids, particularly in monogalactosyldiacylglycerol, phosphatidylcholine and phosphatidylglycerol. This fatty acid was exclusively located at the sn-1 position of the glycerol moiety in all polar lipids except for phosphatidylcholine. In phosphatidylcholine 20:5 was distributed at both the sn-1 and sn-2 positions. 16:3 was concentrated at the sn2 position of monogalactosyldiacylglycerol and trans-16:1 (n-13) was dominant at the sn-2 position of phosphatidylglycerol. C₁₈ fatty acids, the minor fatty acids in P. tricornutum, were confined to the sn-2 position of phosphatidylcholine.     

Non-methylene-interrupted fatty acids from marine invertebrates: Occurrence,characterization and biological properties

Marine organisms, in particular invertebrates, have proved to be a major source of unique fatty acid (FA) structures originating from unusual biosynthetic pathways. Among them, non-methylene-interrupted (NMI) FA occur in various molluscs in the wide ranges of concentrations (up to 20%), such as the most often encountered 20:2 Δ5,11, 20:2 Δ5,13, 22:2 Δ7,13 or 22:2 Δ7,15. Such NMI FA have also been reported from algae, echinoderms, sponges, tropical rays, and many other invertebrates. The most intriguing marine invertebrates seem to be sponges that commonly contain very long-chain Δ5,9 FA. A third double bond can occur in the NMI FA as reported in some marine organisms, such as 20:3 Δ7,13,16 or 30:3 Δ5,9,23. Lipids of invertebrates from deep-sea hydrothermal and cold-seep vents gave rise to an intense research activity including reports on unprecedented NMI polyunsaturated FA. The bivalve molluscs are able to synthesize de novo the NMI FA but their precise biological interest is presently not well-known, although structural and functional roles in biological membranes have been suggested, in particular a higher resistance to oxidative processes and microbial lipases. Biosynthetic pathways of Δ5,9 FA in sponges were demonstrated up to C₂₆ FA structures and include particular elongation and desaturation steps. Recently, intense research effort has been conducted to investigate the biomedical potential of these unusual FA. Thus, Δ5,9 FA displayed interesting antiplasmodial activity. The most promising FA topoisomerase I inhibitors to date seem to be the long-chain Δ5,9 FA. This inhibitory activity is probably partially responsible for the toxicity displayed by some of the Δ5,9 FA towards cancer cell lines.     

Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.     

ACL-I, a lectin from the marine sponge Axinella corrugata: isolation, characterization and chemotactic activity

The lectin from the marine sponge Axinella corrugata (ACL-I) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-I is a hexameric glycoprotein with a Mr of 82.3 kDa estimated by SDS-PAGE and 78.5 kDa by FPLC on Superose 12 HR column. The pI of lectin is 6.3 and ACL-I is constituted of 13.9 kDa similar subunits some of them linked by disulphide bridges. This lectin agglutinates native rabbit, goat and dog erythrocytes and in less extent human erythrocytes. The hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but it is strongly inhibited by carbohydrates containing N-acetyl groups. ACL-I is stable up to 70 degrees C for 30 min, with optimum pH between 7 and 8, and it is also resistant to enzymatic proteolysis in vitro. In the presence of reducing or denaturant agents, the lectin activity decreases. ACL-I displays chemotactic effect on rat neutrophil in vitro which is inhibited by N-acetyl-d-glucosamine.     

Identification and characterization of lipids from endosomes purified by electromagnetic chromatography

Traditional separation techniques do not yield endolysosomes of sufficient purity to permit detailed biochemical characterization of this important class of intracellular vesicles. Here, we have used a magnetic chromatography technique to isolate the endosomes from rat peritoneal macrophages and studied their lipid composition. Electromagnetic isolation works by retention of colloidal iron containing vesicles on magnetic column. The data suggested that both early and late endosomes were rich in cholesterol, whereas sphingomyelin (SM) and specific phospholipids like phosphatidylcholine. phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine are enriched in the late compartments. Our results also indicated that the purified fractions are enriched in raft lipids like SM, but not in cholesterol. The endosomal purification method described here yields pure endosomes with little or no contamination from mitochondria and hence could be used for further biochemical and marker analysis, giving insight into mechanisms of endocytic traffic.     

Chitin catabolism in the marine bacterium Vibrio furnissii. Identification, molecular cloning, and characterization of A N, N'-diacetylchitobiose phosphorylase

The major product of bacterial chitinases is N,N'-diacetylchitobiose or (GlcNAc)(2). We have previously demonstrated that (GlcNAc)(2) is taken up unchanged by a specific permease in Vibrio furnissii (unlike Escherichia coli). It is generally held that marine Vibrios further metabolize cytoplasmic (GlcNAc)(2) by hydrolyzing it to two GlcNAcs (i.e. a "chitobiase "). Here we report instead that V. furnissii expresses a novel phosphorylase. The gene, chbP, was cloned into E. coli; the enzyme, ChbP, was purified to apparent homogeneity, and characterized kinetically. The DNA sequence indicates that chbP encodes an 89-kDa protein. The enzymatic reaction was characterized as follows. (GlcNAc)(2)+P(i) GlcNAc-alpha-1-P+GlcNAc K'(cq)=1.0+/-0.2 Reaction 1 The K(m) values for the four substrates were in the range 0.3-1 mm. p-Nitrophenyl-(GlcNAc)(2) was cleaved at 8.5% the rate of (GlcNAc)(2), and p-nitrophenyl (PNP)-GlcNAc was 36% as active as GlcNAc in the reverse direction. All other compounds tested displayed 相似文献