Induction of non-bilayer lipid structures by functional signal peptides.

Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.

Effects of fish oil supplementation in rheumatoid arthritis.

Sixteen patients with rheumatoid arthritis entered a trial to determine the clinical and biochemical effects of dietary supplementation with fractionated fish oil fatty acids. A randomised, double blind, placebo controlled crossover design with 12 week treatment periods was used. Treatment with non-steroidal anti-inflammatory drugs and with disease modifying drugs was continued throughout the study. Placebo consisted of fractionated coconut oil. The following results favoured fish oil rather than placebo: joint swelling index and duration of early morning stiffness. Other clinical indices improved but did not reach statistical significance. During fish oil supplementation relative amounts of eicosapentaenoic acid and docosahexaenoic acid in the plasma cholesterol ester and neutrophil membrane phospholipid fractions increased, mainly at the expense of the omega-6 fatty acids. The mean neutrophil leucotriene B4 production in vitro showed a reduction after 12 weeks of fish oil supplementation. Leucotriene B5 production, which could not be detected either in the control or in the placebo period, rose to substantial quantities during fish oil treatment. This study shows that dietary fish oil supplementation is effective in suppressing clinical symptoms of rheumatoid arthritis.

Maturation-associated loss and incomplete de novo synthesis of the transferrin receptor in peripheral sheep reticulocytes: response to heme and iron.

Hemin, but not iron, in the culture medium stimulates the maturation-associated loss of the transferrin receptor from sheep reticulocytes (t1/2 for loss approximately 6 hr) and its appearance in a population of externalized vesicles. A similar pattern is seen with nucleoside binding (a measure of the nucleoside transporter), where hemin increases the loss of binding activity from the cells during culture, concomitant with an increase in nucleoside binding in the externalized vesicles. Sheep reticulocytes retain the ability to synthesize the transferrin receptor, but the 35S-labeled receptors are not detected in released vesicles. Whereas hemin stimulates the loss of 35S-labeled transferrin receptors from the cell (t1/2 for loss approximately 20 hr), nonheme iron is more effective than heme. This difference in response of native and 35S-labeled receptor to hemin and iron supplements appears to be related to the differences in the two classes of receptors. Although the 35S-labeled receptor binds transferrin and both native and 35S-labeled peptides comigrate after chemical deglycosylation, the 35S-receptor is approximately 2 kD smaller than the native receptor and fails to acquire its complete size even when chased for up to 24 hr. Moreover, the 35S-labeled receptor is not expressed at the cell surface, but is retained in a nonrecycling compartment, where it is insensitive to digestion by trypsin at both 0 degrees C and 37 degrees C.

Ultrastructural demonstration of increased sulfated proteoglycans and calcium associated with chondrocyte cytoplasmic processes and matrix vesicles in rat growth plate cartilage.

A monoclonal antibody (3-B-3) to chondroitin 6-sulfated proteoglycan was used with immunoperoxidase electron microscopy to study the relationship of chondrocyte cytoplasmic processes and matrix vesicles in rat epiphyseal growth plate cartilage. Immunoperoxidase staining of the chondrocyte plasmalemma was found at all levels in the growth plate and was most prominent in the hypertrophic zone. The plasmalemma and matrix of the cytoplasmic process often demonstrated stronger reactivity than the remainder of the cell surface. Matrix vesicles showed weak to strong surface or internal reactivity. The majority of them stained very similarly to the cytoplasmic process. X-ray microanalysis of specimens processed by rapid freezing and freeze substitution confirmed that both sulfur and calcium were localized within or in close association with both the cytoplasmic process and the matrix vesicle, suggesting a chemical combination of calcium with sulfated proteoglycans at both sites. These results indicate that there is a selective increase in the concentration of membrane-associated sulfated proteoglycan and calcium in the cell process, from which matrix vesicles may be released into the extracellular matrix.

Effect of dietary omega-3 and omega-6 fatty acids on development of azaserine-induced preneoplastic lesions in rat pancreas.

We examined the effect of varying the ratio of dietary omega-3 (omega 3) to omega-6 (omega 6) on the development of pancreatic preneoplastic lesions in male Wistar rats given azaserine at 14 days of age. As the ratio of dietary omega 3 to omega 6 fatty acids increased in a diet totaling 20% by weight of fat, the development of preneoplastic atypical acinar cell nodules (AACNs) at 4 months after dosing with azaserine decreased significantly. In addition, serum levels of prostaglandin thromboxane B2, prostaglandin E2, and 6-keto-prostaglandin F1 alpha decreased significantly. The fatty acid composition of the rbc membrane was also significantly influenced by the ratio of dietary omega 3 to omega 6 fatty acids. In a second experiment, we examined the effect of dietary intervention with a different type of fat (corn oil or menhaden oil) 2 months into the 4-month postdosing period on AACN development at the end of the post-dosing period. Intervention of the omega 6 fatty acid-rich diet with the omega 3 fatty acid-rich diet significantly decreased focal development. The opposite was true when intervention involved substituting the omega 3 fatty acid-rich diet with the omega 6 fatty acid-rich diet.

The location of calmodulin in the pea plasma membrane.

Plasma membrane has been prepared from pea seedlings in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Calmodulin has been detected in these plasma membrane preparations using calcium overlay techniques, immunoblots, quantitation with antibodies raised against spinach calmodulin, phosphodiesterase activation, mobility shift, and heat stability. EGTA-stable calmodulin represents 0.5-1% of the total plasma membrane protein, and it is the only detectable calcium-binding protein in plasma membrane isolated under these conditions. The anti-spinach calmodulin reacts only with the N-terminal region of spinach calmodulin representing residues 1-106. The positioning of EGTA-stable calmodulin in the plasma membrane has been probed with trypsin and anti-spinach calmodulin. The data suggest that the calmodulin N-terminal region representing residues 1-106 projects from the membrane and could be available for binding other proteins. Calcium-dependent calmodulin binding to the plasma membrane has also been detected. Calcium-dependent calmodulin-binding proteins have been characterized using calmodulin overlay methods. The exposure of calmodulin-binding domains of most of these proteins from the plasma membrane is further suggested by their reaction with azidoiodinated calmodulin.

Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations.

Paracoccus denitrificans grown in a complex medium was highly susceptible to lysozyme, in contrast to cells grown in a complex medium supplemented with Mg2+ and Ca2+ or in a succinate-salts medium. The complex medium was deficient in divalent cations needed for optimum outer membrane stability. The major change in molecular compositions of outer membranes isolated from cells grown under the different conditions was a higher ratio of ornithine-containing lipid to phospholipid in complex-medium-grown cells (0.63) than in cells grown in complex medium with Mg2+ and Ca2+ (0.22) or in succinate-salts medium (0.14). We suggest that the dipolarionic ornithine-containing lipid is less dependent than acidic phospholipids on divalent cations for its incorporation into the outer membrane.

The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein.

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.

The influence of virus transformation and cell population density on some membrane properties of mouse fibroblasts in culture.

The influence of cell population density and simian virus 40 transformation on the activity of the Na-K pump was studied in mouse fibroblasts cultured in medium supplemented with fetal bovine serum. The activity of the Na-K pump was determined from K+ influx, ethacrynate-sensitive K+ influx, (Na+ + K+)-ATPase assay, and the determinations of intracellular potassium and sodium ion concentrations in these cells. The activity of the Na-K pump was found to decrease in density-inhibited cultures of normal fibroblasts (designated as 3T3 cells), while in the virus-transformed cells (SV3T3) the activity remained fairly constant at all cell population densities.

Determination of selenium in human spermatozoa and prostasomes using base digestion and electrothermal atomic absorption spectrophotometry.

A method for the determination of selenium in human spermatozoa and prostasomes is described. The samples were digested with 25% (w/v) tetramethylammonium hydroxide (TMAH) in methanol and analyzed by atomic absorption spectrometry with electrothermal atomization and Zeeman background correction (ET-AAS). Nickel was used as a matrix modifier. Calibration was performed using the matrix-based calibration curve. The TMAH-digestion method agreed well with a conventional digestion procedure using concentrated nitric acid. The TMAH-digestion does not require heating or strong acids and it was suitable for small biological samples. The average recovery of added selenium in spermatozoan digests was 95.1 +/- 5.2% (n = 5). The coefficient of variation was 9.1% (n = 21). The accuracy of the method tested with the NBS standard 1577 (bovine liver, certified at 1.1 +/- 0.1 micrograms Se/g) resulted in a value of 0.98 +/- 0.10 micrograms Se/g (n = 16). The method was further tested in an interlaboratory comparison study.

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum.

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.

Circadian rhythms in Neurospora crassa: membrane composition of a mutant defective in temperature compensation.

The cel mutant of Neurospora, partially blocked in fatty acid synthesis and lacking temperature compensation of its circadian rhythm below 22 degrees C, had a phospholipid fatty acid composition in liquid shaker culture distinctly different from that of a cel+ control strain. During growth, cel+ exhibited a reproducible increase in its linoleic acid level from about 32 to a plateau at 63 mol%, and a corresponding decrease in its linolenic acid level from about 40 to a plateau at 10 mol%. The level of palmitic acid was constant at 19 mol%. In the cel strain, the linoleic acid level was constant at 54 mol% while the palmitic acid level increased from about 12 to about 23 mol%. Supplementation with palmitic or linoleic acids altered the patterns of fatty acid composition of cel, but did not affect the pattern of cel+. Altered fatty acid composition cosegregated with the cel marker. The mitochondrial phospholipids of cel in liquid culture also had abnormal fatty acid composition, as did the whole mycelial phospholipids on solid medium. These results are consistent with the involvement of membrane homeostasis in the temperature compensation of circadian rhythms.

Heat sensitivity and membrane properties of metastasizing and non-metastasizing rat mammary tumors.

Paired lines of metastasizing and non-metastasizing transplantable rat mammary tumors were studied for their sensitivity to hyperthermia. The metastasizing TMT-081 and SMT-2A tumors were markedly more sensitive to heat injury as measured by tumor growth delay than were their non-metastatic counterparts MT-100 and MT-W9B. The metastasizing MT-081 tumor was also significantly more sensitive than the SMT-2A tumor. The differences in heat sensitivity were not the result of differences in intra-tumor temperatures attained during water bath heating. A more likely explanation for the variable response obtained with these tumors after heat treatment may be the inherent differences in stability and composition of their cellular membranes, particularly the plasma membrane.

Electron probe analysis, X-ray mapping, and electron energy-loss spectroscopy of calcium, magnesium, and monovalent ions in log-phase and in dividing Escherichia coli B cells.

The elemental composition of individual cells of rapidly frozen and cryosectioned Escherichia coli B was measured with electron optical microanalytic methods. The Ca content was high (26.2 mmol/kg) in a 10-nm-wide region of the cell envelope. Amounts of cytoplasmic Ca in actively dividing cells were significantly higher (32.6 mmol/kg [dry weight]) than in the log-phase (1.5 mmol/kg) cells. Cellular Mg was 205 mmol/kg (dry weight) and it was uniformly distributed throughout the cell. Cells washed in distilled water before freezing lost monovalent ions (Na, Cl, and K), but the membrane-bound Ca and cellular Mg were not reduced, indicating that cellular Mg and membrane Ca are more tightly bound.

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function.

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.

Correlated observations and analysis of maturation-ameloblast morphology and enamel mineralization.

A combined HCl-collagenase digestion technique and scanning electron microscopy were used to isolate the enamel organ and to confirm the presence of maturation ameloblasts of both ruffle-ended (RA) and smooth-ended (SA) types on maturing enamel in kitten permanent tooth germs. EDTA perfusion of animals fixed with aldehyde produced two or three belt-like shallow grooves (from 30 to 100 micron wide) running horizontally through the maturing enamel surface, coinciding closely with the SA distribution pattern. In animals that had been perfusion-fixed with unbuffered osmium tetroxide containing 2.5% potassium pyroantimonate, SEM-EDX analysis detected K in a superficial enamel layer overlaid by the SA layer. Potassium concentration decreased gradually toward the deeper layers. Very little K penetrated the enamel under the RA layer. Energy-dispersive x-ray analysis of Ca and P concentrations in the enamel revealed an even distribution of these elements throughout the superficial layer of maturing enamel. These results suggest that the SA layer forms an access route for K and EDTA and that, in spite of the obvious morphological and functional differences between RA and SA, the maturing enamel surfaces overlaid by these two cell types show similar degrees of mineralization.

Dietary effects of trans fatty acids.

The dietary effects of the trans fatty acids in hydrogenated fats in health and disease deserves consideration for at least two reasons. One, trans fatty acids are becoming increasingly important as a source of calories in all industrialized countries, and two, their structural differences from the fatty acids in unhydrogenated fats offer a means of exploring uncharted areas in lipid metabolism. Such an exploration would have been impossible twenty years ago; some areas are still inaccessible because of a lack of methodology.

Lipid alterations in LLC-PK1 cells exposed to mercuric chloride.

We have studied the effects of HgCl2 on the lipids of LLC-PK1 (pig kidney) epithelial cells. Our results show that treatment of cells with HgCl2 caused a rapid accumulation of unesterified fatty acids (particularly arachidonic acid) and lysophospholipids. A 27-fold increase in unesterified arachidonic acid and a 17-fold increase in lysophosphatidylethanolamine (LPE) was accompanied by a 26% decline in the mass of phosphatidylethanolamine as determined by gas chromatography and lipid phosphorus assay. Similar changes were seen following HgCl2 treatment of cells whose lipids were labelled with 14C stearic acid, 3H arachidonic acid, or 14C acetate, but the radiolabelling techniques also identified an increased content of label in lysophosphatidylcholine (LPC) and a corresponding decrease in phosphatidylcholine. These alterations were accompanied by the formation of blebs on the plasma membrane and irreversible injury as indicated by electron microscopy. The possible role of unesterified fatty acids in the pathogenesis of injury was studied by adding fatty acids to the cells. The addition of unsaturated fatty acids (oleic, linoleic, or arachidonic acids) to the cells caused plasma membrane blebbing and loss of viability. Similarly, the addition of LPC or LPE to the cells resulted in cell death; however, plasma membrane blebbing did not result.

Differential responses to adjuvants of macrophages from young virgin, aging virgin and aging breeder mice.

Age related differences in macrophage responsiveness to adjuvants seen previously with thioglycollate induced cells were also found with resident macrophages. Thus, activation of phagocytosis by lipopolysaccharide, polyadenylic-polyuridylic acid complexes and muramyl dipeptides occurred when exposed to macrophages removed from young but not aging C58 and C3H/He mice. However, breeding status differences were observed in that aging virgin mice were unresponsive to these adjuvants, while aging breeder mice responded similarly to young virgin mice. Analysis of any changes in membrane phospholipids was carried out to determine their association with the age related and breeding status differences observed. Significant differences occurred in 32P labelling of phosphatidyl inositol between unstimulated macrophages from young and aging C58 mice. No differences were evident, however, after LPS stimulation. Analysis of macrophage plasma membranes from young and aging mice for cholesterol revealed significant age related differences in their ability to undergo increases in cholesterol content after LPS activation.

Characterization of human uveal melanoma cells by phosphorus 31 nuclear magnetic resonance spectroscopy.

We performed experiments to determine the potential usefulness of nuclear magnetic resonance spectra in the diagnosis and follow-up of ocular melanoma. High-resolution phosphorus 31 nuclear magnetic resonance spectra at 109.3 MHz were obtained for human uveal melanoma, Greene hamster melanoma, and normal human diploid fibroblast cells. Phosphate metabolites were identified and their concentrations were shown to vary among the different cell lines. Uveal melanoma cells contain unusually high concentrations of the phospholipid metabolite phosphorylcholine and the phosphodiesters glycerol 3-phosphoryl choline and glycerol 3-phosphoryl ethanolamine. Baseline data are thus provided for studies of the effect of various treatment modalities on uveal melanoma. These initial results suggest that the data provided by high-resolution phosphorus 31 nuclear magnetic resonance spectra can provide useful diagnostic and follow-up data with respect to ocular melanoma.