An oil-degrading bacterium: Rhodococcus erythropolis strain 3C-9 and its biosurfactants

AIMS: To isolate a biosurfactant-producing bacterium and find new products within its culture. METHODS AND RESULTS: A biosurfactant-producing bacterium identified as Rhodococcus erythropolis (3C-9 strain) was isolated from seaside soil. When n-hexadecane was supplied as the sole carbon source, two types of biosurfactants (free fatty acids and glycolipids) were detected in the supernatant of the bacterial culture by use of thin layer chromatography (TLC). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the former consisted of at least 12 free fatty acids of chain lengths from C(9) to C(22); and the latter contained 2 kinds of glycolipids (a glucolipid and a trehalose lipid), which were detected by use of TLC, as well as GC-MS. The hydrophobic moieties of both glycolipids consisted of seven types of straight-chain fatty acids of varying compositions, with chain lengths ranging from C(10) to C(18). It was also noted that biosurfactants of strain 3C-9 were produced in a manner that was growth-related and cannot be synthesized from water-soluble substrates. The effects to enhance the solubility of polycyclic aromatic hydrocarbons and the degradation rate of hexadecane were also tested. CONCLUSIONS: The biosurfactants produced by strain 3C-9 of R. erythropolis included two kinds of glycolipids, as well as free fatty acids. These biosurfactants were notably different from those of previously reported Rhodococcus species, both in terms of their structure and chemical composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain 3C-9 of R. erythropolis is a competitive candidate for use in oil spill cleanup operations, or in new biosurfactant exploration. The findings in this report show that Rhodococcus is a natural reservoir of new biosurfactants.     

Cyclic glycolipids from glandular trichome exudates of Cerastium glomeratum

Fourteen cyclic glycolipids, named glomerasides A–N, have been isolated from the glandular trichome exudate of Cerastium glomeratum (Caryophyllaceae). Their structures were determined by spectroscopic analysis of the glycolipids, as well as by application of the Ohrui–Akasaka method to the fatty acid methyl esters derived from the glycolipids and GCMS studies of trimethylsilyl ether derivatives of the methyl esters. The various glomerasides have a glycosidic linkage between the anomeric hydroxy group of the glucose and the C-11, C-10 or C-9 positions of the docosanoyl moiety. They also contained an ester linkage between the C-6 hydroxy group of the glucose ring and the carboxyl group of the oxygenated fatty acid to form their macrocyclic structures. The glucose moiety was optionally acetylated and/or malonylated at the C-2 or C-3 hydroxy groups. Among these compounds, the 1,6′-cyclic ester of 11(R)-(2-O-acetyl-β-d-glucopyranosyloxy)docosanoic acid (glomeraside D) was the most abundant (25%).     

The structure and enzymic activities of the C1r and C1s subcomponents of C1, the first component of human serum complement.

The subcomponents C1r and C1s and their activated forms C-1r and C-1s were each found to have mol.wts. in dissociating solvents of about 83000. The amino acid compositions of each were similar, but there were significant differences in the monosaccharide analyses of subcomponents C1r and C1s, whether activated or not. Subcomponents C1r and C1s have only one polypeptide chain, but subcomponents C-1r and C-1s each contain two peptide chains of approx. mol.wts. 56000 ("a" chain) and 27000 ("b" chain). The amino acid analyses of the "a" chains from each activated subcomponent are similar, as are those of the "b" chains. The N-terminal amino acid sequence of 29 residues of the C-1s "a" chain was determined, but the C-1r "a" chain has blocked N-terminal amino acid. The 20 N-terminal residues of both "b" chains are similar, but not identical, and both show obvious homology with other serine proteinases. The difference in polysaccharide content of the subcomponents C-1r and C-1s is most marked in the 'b' chains. When tested on synthetic amino acid esters, subcomponent C-1r hydrolysed both lysine and tyrosine ester bonds, but subcomponent C-1r did not hydrolyse any amino acid esters tested nor any protein substrate except subcomponent C1s. The lysine esterase activity of subcomponent C1s provides a rapid and sensitive assay of the subcomponent.     

Effect of growth temperature on lipid composition of Streptococcus faecium

The effect of growth temperature on the lipid and fatty acid composition of Streptococcus faecium has been studied. No differences in the qualitative composition of S. faecium lipids were observed. In all isolated fractions (neutral lipids, glycolipids, and phospholipids plus other polar lipids), the major fatty acids were palmitic (C-16:0), palmitoleic (C-16:1), octadecenoic (C-18:1), and cyclopropane (C-19:0). Changes in the fatty acid composition of the different fractions were observed which depended on growth temperature; the most significant one was the decrease of octadecenoic acid and the increase of palmitic acid in glycolipids and polar lipids as the temperature increased. The level of cyclopropane C-19:0 was approximately eightfold lower at 8 degrees C than at the other temperatures tested (20, 30, and 45 degrees C).     

Glycolipids of peripheral nerve: isolation and characterization of glycolipids from rabbit sciatic nerve

Besides cerebreside and sulfatide four other glycolipids were isolated from rabbit sciatic nerve and analyzed by chemical and chromatographic methods. Three of the glycolipids were shown to be fatty acid esters of cerebroside; the fourth was characterized as diacyl glycerol galactoside and its alkyl ether analog. In the ester linkage mainly unsubstituted acids with chain length C(16) to C(18) were present. Both hydroxy and unsubstituted acids were found in amide linkage. They varied in chain length from C(16) to C(24) and were typical of cerebrosides. The long-chain base fraction contained sphingosine and dihydrosphingosine as the main components.     

Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics

Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli chi2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42 degrees C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli chi1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of chi1776 or in chi2452 grown at 32 degrees C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of chi2452 grown at 42 degrees C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of chi2452 cells grown at 42 degrees C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of chi2452 grown either at 42 or 32 degrees C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings.     

An intragenic revertant of a poliovirus 2C mutant has an uncoating defect.

A revertant was isolated from a temperature-sensitive poliovirus 2C mutant, 2C-31, which is defective in viral RNA synthesis. This revertant, called 2C-31R1, grew well at 39 degrees C and was not defective in RNA synthesis. However, in contrast to its parental mutant, 2C-31R1 was cold sensitive and could hardly grow at all at 32 degrees C. Analysis of a single-cycle growth revealed that 2C-31R1 was defective in virion uncoating at 32 degrees C, and a substantial amount (more than 30%) of input viruses could be recovered as infectious particles from an infected cell lysate up to 6 h postinfection. The uncoating defect and the inability to grow at cold temperatures could be overcome by a brief incubation at the permissive temperature (39 degrees C) before the infection was continued at 32 degrees C. cDNA cloning and mix-and-match recombination experiments indicated that the defect in uncoating was the result of two secondary point mutations, seven nucleotides apart, in the 2C-coding sequence downstream of the inserted linker which is the original mutation in the parental 2C-31 genome. Another revertant, 2C-31R3, isolated from the same 2C-31 stock, was not defective in uncoating and appeared to be a secondary revertant that contained an intragenic suppressor for the uncoating defect. The uncoating defect of 2C-31R1 could be complemented by type 2 poliovirus. These results suggested that protein 2C, in addition to its role in viral RNA synthesis, has a function in determining virion structure.     

The crucial role of trehalose and structurally related oligosaccharides in the biosynthesis and transfer of mycolic acids in Corynebacterineae

Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.     

A family of glycoinositol phospholipids from Leishmania major. Isolation, characterization, and antigenicity

The glycolipids of the protozoan Leishmania major strain LRC-L119 belong to a class of glycoinositol phospholipids (GIPL) that show partial structural homology to the phosphatidylinositol-containing glycolipid membrane anchors of several eukaryotic proteins and the lipid moiety of L. major lipophosphoglycan. The GIPLs were the only glycolipids detected and were purified by octyl-Sepharose and thin layer chromatographies. Analysis of the native and dephosphorylated glycolipids (GIPLs 1-6) by gas chromatography-mass spectrometry revealed that the glycan moieties have between 4 and 10 saccharide residues and all contain mannose, galactose, and non-N-acetylated glucosamine. Some of the GIPLs also contain glucose (GIPL-6) and hexose monophosphate residues (GIPL 4-6). The presence of an inositol phospholipid moiety in all the GIPLs is indicated by the identification of 1 myo-inositol monophosphate residue/molecule and their susceptibility to phosphatidylinositol-specific phospholipase C. However, heterogeneity in the lipid moieties is indicated by differences in the compositional analysis and the behavior of the GIPLs on the thin layer chromatography after mild alkali hydrolysis or phospholipase A2 treatment. These results demonstrate that GIPLs 1-4 contain 1-alkyl-2-acylglycerol composed of saturated unbranched alkyl chains with carbon chain lengths of 18-26 and acyl chains of myristate, palmitate and stearate, whereas GIPL-5 and -6 contain lyso-alkylglycerol composed of mainly C24:0 and C26:0 alkyl chains. Analysis of the products of nitrous acid deamination demonstrates that these glycerolipids are present as alkylacylphosphatidylinositol (GIPLs 1-4) and 1-O-alkylglycerophosphoinositol (GIPL-5 and -6), respectively. GIPL-2 and -3 are labeled on the surface of living promastigotes with galactose oxidase/NaB[3H]4. These GIPLs also react with three monoclonal antibodies that recognize the surface of promastigotes and amastigotes of L. major and other Leishmania spp.     

Parallel antitumor,granuloma-forming and tumor-necrosis-factor-priming activities of mycoloyl glycolipids fromNocardia rubra that differ in carbohydrate moiety: Structure—activity relationships

Summary Multiple intravenous injections (30 µg, ten times) in ICR mice of trehalose dimycolate and glucose monomycolate fromNocardia rubra, containing C_36–48 mycolic acids, showed a prominent antitumor effect on a subcutaneously implanted sarcoma-180, an allogeneic sarcoma of mice with a significant granuloma formation in lungs, spleen and liver. On the other hand, mycoloyl glycolipids other than glucose monomycolate and trehalose dimycolate, such as mannose or fructose mycolate, showed no significant activity for tumor regression or granuloma formation in mice.Trehalose dimycolate and glucose monomycolate fromN. rubra, and glucose monomycolate with C_56–60 mycolic acids fromRhodococcus terrae also showed a distinctive priming activity for tumor necrosis factor (TNF), when lipopolysaccharide fromEscherichia coli was administered as an eliciting agent. The TNF activity in the sera of mice was abrogated almost completely by anti-(murine TNF) antibody with protein-A—agarose. Again in contrast, mannose and fructose mycolate fromN. rubra and glucose monomycolate with C_30–34 mycolic acids fromRhodococcus equi did not show such activities in mice.Meth-A, a syngeneic fibrosarcoma of BALB/c mice, was less sensitive to administration of glycolipids than sarcoma-180. These results indicated that the existence of a glucose or trehalose molecule was necessary for the expression of immunomodifying activities among various mycoloyl glycolipids differing in carbohydrate structure. However, since the administration of lipopolysaccharide was essentially required as an eliciting agent for the induction of TNF, while no eliciting agent was required for the antitumor activities, TNF does not seem to contribute directly to the antitumor activities of mycoloyl glycolipids in our systems. There was, however, a parallel structure-activity relationship among granuloma-forming, antitumor and TNF-priming activities, indicating that the structures of both the carbohydrate moiety and the mycoloyl residues influenced an initial step, such as macrophage activation, commonly and profoundly.     

Kinetics of folding of alpha alpha-tropomyosin subsequences.

The kinetics of folding random coils of alpha alpha-tropomyson (Tm) subsequences to two-chain coiled coils was studied by stopped-flow CD. Subsequences studied were those comprising residues 11-127 (11Tm127), 142-281 (142Tm281), 1-189 (1Tm189), and 190-284 (190Tm284) of the parent 284-residue alpha-tropomyosin chain. Unlike the parent, subsequences 1Tm189 and 11Tm127 fold within the dead time of the instrument (less than 0.04 s). Like the parent, subsequences 142Tm281 and 190Tm284 fold in two phases. In the fast phase, 45% and 32%, respectively, of the equilibrium helical content form. In the time-resolvable, first-order slow phase (k-1 = 2.7 s at 20 degrees C for 142Tm281 and k-1 = 2.0 s at 15 degrees C for 190Tm284), the remaining structure forms. Neither reduced 142Tm281 nor 190Tm284 show any dependence of the rate on concentration, so chain association occurs in the fast phase. Like the parent 142Tm281 forms more helical content in the fast phase when cross-linked at C-190, and the remaining structure forms slowly with rate parameters similar to those of the reduced species. Comparison of the folding behavior of C- and N-terminal subsequences with that of the parent protein suggests that the slow phase in the parent is caused by a folding bottleneck somewhere nearer the C-terminus. However, rapid association and partial folding near the N-terminus is not necessary for prompt folding, since even 190Tm284 chains associate and partially fold very rapidly (less than 0.04 s), and then complete the folding in seconds.     

Structural determination of the polar glycoglycerolipids from thermophilic bacteria Meiothermus taiwanensis.

The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA-400 by ethanol extraction and purified by Sephadex LH-20 and silica gel column chromatography. The fatty acid composition of O-acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C(15:0) (34.0%) and C(17:0) (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O-acyl groups under mild basic conditions provided two glycolipids, which differ only in N-acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C(17:0) N-acyl group and the other hydroxy C(17:0) in a ratio of about 1 : 3.5. Furthermore, complete de-lipidation with strong base followed by selective N-acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA-400 was determined with the following structure: alpha-Galp(1-6)-beta-Galp(1-6)-beta-GalNAcyl(1,2)-alpha-Glc(1,1)-Gro diester, where N-acyl is C(17:0) or hydroxy C(17:0) fatty acid and the glycerol esters were mainly iso- and anteisobranched C(15:0) and C(17:0).     

Thermotropic properties and molecular dynamics of cholesteryl ester rich very low density lipoproteins: effect of hydrophobic core on polar surface

Cholesteryl ester rich very low density lipoproteins (CER-VLDL), isolated from the plasma of rabbits fed a hypercholesterolemic diet, have been studied by differential scanning calorimetry (DSC), 13C nuclear magnetic resonance (NMR), and spin-label electron paramagnetic resonance (EPR) to determine the temperature-dependent dynamics of cholesteryl esters in the hydrophobic core and of phospholipids on the polar surface. Intact CER-VLDL exhibit two DSC heating endotherms; these occur at 40-42 and 45-48 degrees C. Cholesteryl esters isolated from CER-VLDL also exhibit two DSC endotherms; these occur at 50.0 and 55.1 degrees C and correspond to the smectic----cholesteric and cholesteric----isotropic liquid-crystalline phase transitions. A model mixture containing cholesteryl linoleate, oleate, and palmitate in a ratio (0.21, 0.51, and 0.28 mol fraction) similar to that in CER-VLDL exhibited comparable DSC endotherms at 45.2 and 51.5 degrees C. CER-VLDL at 37 degrees C gave 13C NMR spectra that contained no resonances assignable to cholesteryl ring carbons but detectable broad resonances for some fatty acyl chain carbons, suggesting the cholesteryl esters were in a liquid-crystalline state. When the mixture was heated to 42 degrees C, broad ring carbon resonances became detectable; at 48 degrees C, they became narrow, indicating the cholesteryl esters were in an isotropic, liquid-like state. With increasing temperature over the range 38-60 degrees C, the resonances for cholesteryl ring carbons C3 and C6 in CER-VLDL narrowed differentially. Similar spectral changes were observed for the synthetic cholesteryl ester mixture, except they occurred at temperatures about 10 degrees C higher. These results indicate that the two DSC transitions in CER-VLDL do not directly correlate with the smectic----cholesteric and cholesteric----isotropic transitions exhibited by pure cholesteryl esters. (5-Doxylpalmitoyl)-phosphatidylcholine (5-DP-PC) and (12-doxylstearoyl)phosphatidylcholine (12-DS-PC) were used to probe the polar surface monolayer of CER-VLDL; the corresponding cholesteryl esters (5-DP-CE and 12-DS-CE) were used to probe the hydrophobic core. None of these probes in CER-VLDL detected an abrupt change in EPR order parameters, S, or maximum splitting, 2T max, over the temperature range 20-58 degrees C even though 12-DS-PC and 5-DP-PC can detect phase transitions in phospholipid bilayers and 12-DS-CE and 5-DP-CE can detect phase transitions in neat cholesteryl esters. However, 12-DS-CE and 5-DP-CE did detect a much greater acyl chain order for the neutral lipids of CER-VLDL than for those of normal triglyceride-rich VLDL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and fine-structure characterization of four monoclonal antibodies reactive with glycoconjugates containing terminal GlcNAc residues: application to aspects of lacto-series tumor antigen biosynthesis.

A series of murine monoclonal antibodies, each reactive with terminal GlcNAc residues expressed on glycolipids, have been isolated after immunization with the glycolipid nLc5 (GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1---- 4Glc beta 1----1Cer). The derived antibodies, designated TE-4, TE-5, TE-6, and TE-7, were tested for binding specificity with a variety of terminal GlcNAc-containing oligosaccharides expressed on glycolipids and glycoproteins. Antibody TE-4 was found to be reactive only with linear and branched terminal GlcNAc beta 1----3Gal containing structures present in lacto-series carbohydrates irrespective of core chain length. The binding specificity of TE-7 was similar except that no reactivity was observed with the short chain structure Lc3 and was weakly reactive with branched agalacto-I structures, suggesting a longer recognition epitope than for the TE-4 antibody. Antibodies TE-5 and TE-6 reacted with terminal GlcNAc beta 1----3Gal structures and as well GlcNAc beta 1----2(6)Man structures present on BSA-oligosaccharide conjugates. Weak binding was also observed with GlcNAc beta 1----6Gal structures with these antibodies. TE-5 was found to be particularly sensitive to low amounts of terminal GlcNAc-containing glycolipids in both solid phase assays and in TLC-immunostaining studies of neutral glycolipids extracted from colonic adenocarcinoma cell lines and tumors. No reactivity was observed with internal GlcNAc residues with any antibody tested. The panel of antibodies was applied to studies of binding to Triton X-100-solubilized fractions from normal mucosal and adenocarcinoma cell lines after desialylation and Smith degradation to expose terminal GlcNAc residues on glycoproteins and glycolipids. Binding of antibodies TE-4 and TE-7 was restricted to adenocarcinoma-derived cell fractions. Application of these antibodies in studies of lacto-series core chain synthesis and in immunodiagnostic procedures after initial treatments to concentrate lacto-series antigens into terminal GlcNAc-containing structures is discussed.     

The specific glycosphingolipid composition of human ureteral epithelial cells

Total non-acid and acid glycolipid fractions were isolated from epithelial cell scrapings and the non-epithelial residue of a human upper ureter. The glycolipid fractions were structurally characterized as total mixtures by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy. Selected structural information was also obtained on binding of monoclonal antibodies and bacteria to the thin-layer chromatograms. The major epithelial cell glycolipids were Glc beta 1-1ceramide (75%), dihexosylceramide (10%) and NeuAcLacceramide (10%). In addition, 8 minor glycolipids belonging to the blood group P, Lewis and ABO systems were identified. The major glycolipids of the non-epithelial residues were mono- and dihexosylceramides together with globotriaosyl- and globotetraosylceramides. The epithelial mono- and diglycosylceramide compounds had an unusual ceramide composition with mainly C18 and C20 trihydroxy long chain bases in combination with C22-C24 hydroxy fatty acids in contrast to the non-epithelial glycolipids which contained mainly C18 dihydroxy long chain bases in combination with C16-C24 non-hydroxy fatty acids.     

Structure and properties of mixed-chain phosphatidylcholine bilayers

The structural and thermotropic properties of the hydrated mixed-chain phosphatidylcholines (PCs), C(8):C(18)-PC and C(10):C(18)-PC, have been studied by X-ray diffraction and differential scanning calorimetry. For fully hydrated C(8):C(18)-PC, the reversible chain melting transition is observed at 9.9 degrees C (delta H = 7.3 kcal/mol). X-ray diffraction at 0 degrees C (below the chain melting transition) shows a small bilayer repeat distance, d = 51.0 A, and a sharp, symmetric wide-angle reflection at 4.1 A, characteristic of a mixed interdigitated bilayer gel phase [see McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038-4044; Hui, S. W., Mason, J. T., & Huang, C. (1984) Biochemistry 23, 5570-5577]. At 30 degrees C (above the chain melting transition), a diffuse band is observed at 4.5 A characteristic of an L alpha phase but with an increased bilayer periodicity, d = 61 A. Both the calculated lipid bilayer thickness (d1) and that determined directly from electron density profiles (dp-p) show unusual increases as a consequence of chain melting. In contrast, fully hydrated C(10):C(18)-PC shows an asymmetric endothermic transition at 11.8 degrees C. Below the chain melting transition, two lamellar phases are present, corresponding to coexisting interdigitated (d = 52.3 A) and noninterdigitated (d = 62.5 A) bilayer gel phases. The relative amounts of these phases depend upon the low-temperature incubation and/or hydration conditions, suggesting conversions, albeit kinetically complex, between metastable, and stable phases. The different behavior of C(8):C(18)-PC and C(10):C(18)-PC, as well as their positional isomers, is rationalized in terms of the molecular conformation of PC.     

Differential scanning calorimetry of light meromyosin fragments having various lengths of carp fast skeletal muscle isoforms

Various recombinant light meromyosin (LMM) fragments were prepared from cDNAs encoding the 10 degrees C and 30 degrees C types of myosin heavy chain isoforms predominantly expressed in fast skeletal muscles of the 10 degrees C- and 30 degrees C-acclimated carp, respectively. These included three kinds of quarter fragments, 1/4-, 2/4-, and 4/4-quarter, composed of residues 1-130, 131-270, and 401-563 from the N-terminus, respectively, as well as three halves, N-, M-, and C-half fragments, containing residues 1-301, 131-400, and 302-563, respectively, and 69K fragments of residues 1-525. Unfortunately, in spite of extensive efforts, the 3/4-quarter fragment was not expressed for both 10 degrees C and 30 degrees C types in our expression system using Escherichia coli. All the LMM fragments except for the 10- and 30-2/4 quarters for the 10 degrees C and 30 degrees C types, respectively, exhibited a typical pattern of a-helix in CD spectrometry. When these were subjected to differential scanning calorimetry (DSC), 30 degrees C-type LMM fragments were all found to be more thermostable than the 10 degrees C-type counterparts. To identify amino acid substitutions responsible for different thermostabilities between the 10 degrees C- and 30 degrees C-type LMMs, six mutant proteins were prepared, mainly focusing on substitutions in the C-terminal half of LMM, and subjected to DSC and CD analyses. For three mutants in which two residues of the 10 degrees C type were replaced by those of the 30 degrees C type, 10-S355T/T361A, 10-M415L/L417V, and 10-S535A/H536Q, the endothermic peaks in DSC increased by 1.4-2.0 degrees C from that of the original 10 degrees C type. The T(m) values for two single-residue substitutions, 10-H449R and 10-T491I, shifted 0.8 and 1.3 degrees C higher than that for the 10 degrees C-type LMM, respectively, whereas the last mutant, 10-G61V, showed no change in thermostability. The finding that the difference in T(m) values for major endothermic peaks from the 10-69K and 30-69K fragments was 4.6 degrees C, which roughly corresponds to that between the original 10 degrees C and 30 degrees C types, suggested that the eight substitutions located in the C-terminal region of the 69K fragments (residues 302-525) are major candidates for the residues responsible for the difference in thermostability between the 10 degrees C- and 30 degrees C-type LMMs.     

Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.     

Determination of the primary and fine structures of galactomannan from seeds of Gleditsia triacanthos f. intermis L

This was the first study to isolate galactomannan, a 660-kDa polysaccharide, from the seeds of Gleditsia triacanthos f. inermis L. (yield 15.4%). Its aqueous solutions were optically active ([alpha] D = +31.0 degrees) and highly viscous ([eta] = 578 ml/g). The analysis of this heteropolysaccharide using chemical, enzymatic, and chromatographic procedures, as well as IR- and 13C-NMR spectroscopy, showed that is consists of D-mannopyranose and D-galactopyranose residues (molar ratio 2.42:1). Its main chain is comprised of 1,4-beta-D-mannopyranose residues, 41% of which is substituted at C-6 with single residues of alpha-D-galactopyranose. The probability of occurrence of differently substituted mannobiose units in the chain, determined experimentally, was 0.16 for the unit Man-Man, 0.50 for the units Gal(Man-Man) and (Man-Man)Gal, and 0.34 for the dissubstitued unit Gal(Man-Man)Gal.     

Effect of fatty acid chain length on initial reaction rates and regioselectivity of lipase-catalysed esterification of disaccharides

In a reaction medium mixture of 9:11 t-BuOH and pyridine (v/v) the effect of fatty acid chain length (C-4-C-12) on C. antarctica lipase B (Novozym 435, EC 3.1.1.3) catalysed esterification was studied. alpha and beta maltose 6'-O-acyl esters in an anomeric molar ratio of 1.0:1.1 were synthesised independently of the chain length, but the initial specific reaction rate increased with decreasing chain length of the acyl donor. The product yield followed the same trend with a lauryl ester yield of 1.1% (mol/mol) and a butyl ester yield of 27.6% (mol/mol) after 24 h of reaction. With sucrose as the acyl acceptor the 6'-O-acyl and 6-O-acyl monoesters were formed with fatty acids of chain length C-4 and C-10 while the 6',6-O-acyl diester was formed only with butanoic acid (C-4:0) as acyl donor. The 6'-O-acyl and 6-O-acyl monoesters and the 6',6-O-acyl diester of butanoic acid were produced in a molar ratio of 1.0:0.5:0.2 and with decanoic acid (C-10:0) the 6'-O-acyl and 6-O-acyl monoesters were formed in the ratio of 1.0:0.3. The highest initial reaction rate and yield were obtained with the shortest chain length of the acyl donor. Initial reaction rates and ester yields were affected by the solubility of the disaccharide, with higher reaction rates and yields with maltose than with sucrose, while no formation of esters were observed with either cellobiose or lactose as acyl acceptors.