Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

Further aspects of IL-1 beta secretion revealed by transfected monkey kidney cells.

Because the cytokine interleukin-1 beta (IL-1 beta) lacks a classical hydrophobic signal sequence, it has been unclear how it is released from cells, and whether release proceeds via a novel mechanism or through non-specific leakage. To address this issue, we have examined the secretion of the recombinant forms of human IL-1 beta from COS monkey kidney cells, which express low levels of endogenous IL-1 beta. Four proteins were expressed: precursor and mature IL-1 beta and precursor and mature IL-1 beta fused to an amino terminal hydrophobic signal sequence from human tissue plasminogen activator. By monitoring the appearance of a known cytosolic protein (ATP citrate lyase) in the medium, we find that the unmodified IL-1 beta s are non-specifically released in very small quantities from the cytosol. On the other hand, the signal sequence-modified IL-1 beta s are glycosylated and efficiently secreted by the ER/Golgi pathway. The secreted, modified-mature protein is also biologically active, suggesting that this pathway has been bypassed for reasons other than maintaining the structural integrity of IL-1 beta. More likely the alternative pathway is a critical aspect of IL-1 biology. The differences in kinetics and quantity of IL-1 beta release from monocytic and COS cells suggest that COS cells lack critical components for the rapid release seen in monocytes.     

Southern analysis of BT-R 1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210?kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp. berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210?kDa protein, termed BT-R₁ (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R ₁ , the gene encoding the 210?kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R₁ as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R₁. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R₁, whereas the other is a closely related homologue.     

Influence of duodenal secretions and its components on release and activities of human brush-border enzymes

The in vitro effects of human duodenal secretions and various combinations of its components on activity and release of enzymes from the human brush border were examined. Sucrase retained activity for 90 min in duodenal secretions, and maltase was almost as stable; lactase lost activity rapidly and alkaline phosphatase was of intermediate stability. Inactivation of lactase could only be partly (50%) attributed to luminal proteases, bile salts and phospholipids played no role. Rate of release of an enzyme from the brush border bore no relationship to its rate of inactivation. When individual proteases were studied, elastase was the most potent for releasing disaccharidases from the brush border; trypsin was ineffective alone but augmented the effect of elastase. Sucrase and maltase were activated by proteolytic release, but activation was abolished by simultaneous exposure of brush borders to bile salts. Lactase was released and rapidly inactivated by proteinases, while alkaline phosphatase appeared to be inactivated without significant release. These results show that there are significant interactions between luminal factors which have been inapparent when studying them in isolation. Loss of functionally useful enzyme does not follow release of sucrase or maltase from the brush border into the lumen but does follow release of lactase. Study of the susceptibility of lactase to inactivation by luminal factors in the various forms of lactose intolerance is warranted.     

Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia

The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.     

ADAPTATIONS TO CLIFF-NESTING IN SOME ARCTIC GULLS (LARUS)

Various aspects of the breeding of three gulls, Larus thayeri, glaucoides and hyperboreus, which nested in colonies both on cliff ledges and on level ground in the eastern Canadian Arctic, were compared with those of the ground-nesting L. argentatus and the cliff-nesting Rissa tridactyla. As the result of adaptation to cliff ledge nesting, many aspects of the breeding biology of R. tridactyla were strikingly different from those of the ground-nesting European L. argentatus, but the behaviour of L. thayeri, glaucoides and hyperboreus clearly spanned these differences. Cliff-nesting individuals of thayeri and glaucoides were most like Rissa; ground-nesting individuals of these species were most like argentatus. L. thayeri was more like Rissa than was glaucoides. With but few exceptions, both cliff- and ground-nesting individuals of hyperboreus were most like argentatus. The factors responsible for the intra-specific differences between cliff- and ground-nesters of thayeri and glaucoides are not clear. Limited gene exchange between cliff and ground colonies occurs. Because of physical features of the nest, first-laid eggs were more liable to fall from ledges than second or third eggs. L. thayeri and glaucoides have evolved separate mechanisms to cope with this problem. Egg shape was multimodal in thayeri and glaucoides. Long pyriform eggs were less liable to fall from ledges than eggs of other shapes. L. thayeri laid more long pyriform eggs as first eggs than did glaucoides. L. thayeri lost fewer eggs than did glaucoides, but glaucoides replaced all lost eggs while thayeri did not. Delayed follicular atresia provided glaucoides with insurance of egg replacement. In thayeri, accessory follicles were reabsorbed after the first egg was laid; in argentatus, after the second egg, and in glaucoides after the third egg. At the approach of a predator, it was advantageous for cliff-dwelling chicks to remain motionless but for ground-dwelling chicks to flee their nests and to hide. Among the cliff-nesters, the “freezing in place” reaction of chicks was best developed in thayeri, to a lesser extent in glaucoides, and least in hyperboreus. Among the ground-nesters, chicks of glaucoides and hyperboreus behaved like those of argentatus and fled their nests when disturbed, but chicks of thayeri froze like their cliff-dwelling siblings. Reciprocal transfers of eggs and chicks between cliff and ground colonies indicated that in argentatus, glaucoides and hyperboreus, the factors determining a chick's reaction to disturbance came into play between hatching and the eighth day. In thayeri, the reaction appeared to be effectively innate. Chicks of glaucoides showed a greater predisposition to this behaviour than chicks of argentatus after both had received identical experience on cliff ledges. In thayeri, stereotypy of the freezing reaction has probably been a factor limiting the colonisation of areas where cliffs are scarce but predators present. In argentatus, lack of perfection of this behaviour (compared to glaucoides and thayeri) has probably been a factor preventing argentatus from attaining cliff ledges. L. hyperboreus, although nowhere abundant, is a widespread species nesting on level ground and on cliff ledges but lacking the modifications observed in glaucoides and thayeri; this is due to its size and aggressiveness, the fact that it picks nest-sites before glaucoides and thayeri arrive in the colonies, and that on cliffs it chooses the largest and most level ledges.     

Conflicting evidence regarding the transport of alpha-glutamyl-dipeptides by human erythrocytes.

A novel ninhydrin-positive compound, N-methyl-D-aspartic acid, was identified in the muscle extracts of the blood shell, Scapharca broughtonii. This compound is already known to have potent neuroexcitatory activity, inducing hypermotility and strong releasing action of serum luteinizing hormone in mammals. This may be, however, the first finding of N-methyl-D-aspartic acid in natural products.     

Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli

The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods. The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. 116, 165-170], constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5. The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6]. This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane. This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated. The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS)