Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region.

The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation.     

Retinoylation of HL-60 proteins. Comparison to labeling by palmitic and myristic acids

Recent studies suggest that a retinoic acid (RA) nuclear receptor or a retinoylated nuclear protein may be involved in the action of RA. We showed previously (Takahashi, N., and Breitman, T. R. (1989) J. Biol. Chem. 264, 5159-5163) that retinoylation involves the formation of a thioester bond and occurs on protein in newly formed cells and in pre-existing cells. In this study, we saw at least 14 retinoylated proteins in HL-60 cells. Greater than 90% of the retinoylation was associated with the nuclear protein described previously. This protein, partially purified from isolated nuclei, bound to DNA-cellulose and was eluted with NaCl. Retinoylation occurred in HL-60 cells exposed to cycloheximide. Thus, retinoylation resembled palmitoylation, both in the covalent bond and the exchangeable reaction involving preformed protein. These similarities prompted us to compare retinoylation with two other fatty acylations in growing HL-60 cells. We found that the major retinoylated protein was labeled by either radioactive palmitic acid or myristic acid. The extent of [3H]palmitic acid labeling of this protein was not reduced by growth in the presence of RA. The extent of retinoylation of this protein was not reduced by growth in the presence of increasing concentrations of palmitic acid. These results raise the possibility that the same protein is a substrate for retinoylation, palmitoylation, and myristoylation.     

Acylation of proteins by myristic acid in isolated mitochondria

Isolated and highly purified mitochondria from rat liver were incubated with [1-14C]myristate, solubilized in boiling sodium dodecyl sulfate, and analyzed by polyacrylamide gel electrophoresis and autoradiography. Six to eight protein bands were found to be radioactively labeled. If the mitochondria were heated for 5 min at 95 degrees C prior to incubation with this fatty acid, no labeling was observed. By preexposing the mitochondria to unlabeled fatty acids of varying chain lengths, the extent of labeling by [1-14C]myristate was reduced in a chain length-dependent manner, exhibiting maximal inhibition at lauric acid. Reversibility of the labeling was demonstrated by chasing the incorporated radioactivity with unlabeled fatty acids of varying chain length, resulting in a maximal displacement of the tracer again by lauric acid. Fractionation of the labeled mitochondria into mitochondrial matrix and inner mitochondrial membrane components before or after labeling showed that the modified proteins are located inside the inner mitochondrial membrane. In both cases, the pattern of labeling was different from the one observed with intact mitochondria. The labeled bands in the gel were sensitive to alkaline methanol or hydroxylamine treatment. The radioactivity recovered after this treatment co-migrated with myristic acid on thin layer chromatography plates. The chain length specificity and the rapid reversibility of the observed acylation argue for a new type of reaction, different from the acylation observed in whole cells. The possible involvement of the acylated proteins in the regulation of oxidative phosphorylation is discussed.     

Acylation of cell-associated IL-1 by palmitic acid

To determine whether membrane-associated IL-1 is palmitylated, we labeled LPS-activated human monocytes with [3H]palmitic acid. The plasma membranes were isolated, and the membrane proteins extracted and analyzed simultaneously by SDS-PAGE-autoradiography and Western blot analysis from the same gel. When the monocytes were labeled with [3H]palmitate, 23- and 31-kDa bands were visualized, for membrane-associated IL-1 and its precursor, respectively. The 31- and 23-kDa bands were excised from several gels and rehydrated and analyzed again by SDS-PAGE, autoradiography, and Western blot analysis. The 23- and 31-kDa bands appeared again by both methods. To further investigate membrane-associated IL-1 acylation, human monocytes were labeled with [3H]palmitate, the plasma membranes isolated, and the membrane proteins extracted by CHAPS detergent. Immunoprecipitation of isolated membrane proteins using anti-IL-1 antibodies revealed two bands of 23 and 31 kDa after autoradiography. The studies demonstrate that both membrane-associated IL-1 and the IL-1 precursor are acylated with palmitic acid.     

豆蔻酸和棕榈酸诱导茄子根际拮抗菌与黄萎菌数量消长的关系

以气质联用仪(GC-MS)鉴定的嫁接茄与自根茄根系分泌物主要差异成分豆蔻酸和棕榈酸,利用它们的模拟物质研究了其对茄子根际土壤黄萎菌拮抗真菌与细菌数量和根际土壤黄萎菌数量的影响,明确了两模拟成分对茄子根际拮抗真菌和拮抗细菌的诱导作用及对黄萎菌数量抑制效果。同时,进一步分析了茄子根际拮抗真菌与细菌数总和与根际土壤黄萎菌数量的相关性。结果表明豆蔻酸和棕榈酸处理,茄子根际拮抗真菌和细菌数量均有所增加,在中高浓度下拮抗真菌和细菌数量增加显著。两物质处理下拮抗真菌与细菌数量在开花期和结果期增加显著。两物质对茄子根际黄萎菌数量的影响,整体来说,随两模拟组分浓度的增加土壤黄萎菌数量有所减少,各时期表现有所差异,在开花期两物质处理显著减少了黄萎菌数量。相关性分析表明豆蔻酸和棕榈酸处理下茄子根际拮抗真菌与细菌总量与根际黄萎菌数量呈显著的负相关性。说明嫁接通过改变茄子根系分泌物成分与对茄子根际土壤黄萎菌拮抗菌的诱导有一定关系。     

Acylation of bovine rhodopsin by [3H]palmitic acid

Bovine retinas or preparations of rod outer segments incorporate [3H]palmitic acid into rhodopsin. The incorporation is both time- and temperature-dependent. The major product retains the chromatographic and electrophoretic properties of rhodopsin and remains photosensitive as demonstrated by alteration of its chromatographic behavior upon exposure to light. The incorporated radioactivity resists extraction with organic solvents and is not dissociated from the protein by detergents or under the denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive free fatty acid can, however, be released by alkaline hydrolysis. Hydroxylamine treatment yields a mixture of the free fatty acid and the fatty acyl hydroxamate. These results demonstrate the formation of an ester bond between [3H]palmitic acid and rhodopsin. Cycloheximide fails to inhibit the incorporation. This finding along with the ability of rod outer segments to support the incorporation point to the acylation of rhodopsin as a late post-translational event.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: during the differentiation of keratinocytes, during pregnancy, being evident principally in the endometrium but detectable elsewhere as well, and during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Abstract. Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: (1) during the differentiation of keratinocytes, (2) during pregnancy. being evident principally in the endometrium but detectable elsewhere as well, and (3) during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.     

Formation of acids shorter than palmitic by rat liver cytosol

Particle-free liver supernatant of rats maintained on stock diet synthesizes fatty acids of average chain lengths 9–11 carbon atoms when the protein concentration is high while palmitic acid is the main product when protein concentration is low. This ability to synthesize acids shorter than palmitic is lost on purification of fatty acid synthetase or by starvation of the rats followed by the ingestion of a high sucrose diet. The results are consistent with the presence of a factor in the cytosol, similar to that in lactating mammary glands, which shortens the chain length of the products of fatty acid synthetase.     

Genetic and immunohistochemical detection of mutations inactivating the keratinocyte transglutaminase in patients with lamellar ichthyosis

Autosomal recessive lamellar ichthyosis is a clinically heterogeneous group of severe congenital keratinization disorders that is characterized by generalized hyperkeratosis and variable erythema. About half of the patients have mutations in the TGM1 gene, which encodes the keratinocyte transglutaminase. Linkage studies have shown that at least two further loci for autosomal recessive lamellar ichthyosis must exist. We present here two patients with lamellar ichthyosis caused by mutations in the TGM1 gene. The first patient is compound heterozygous for the novel missense mutation C53S and the splice mutation A3447G. The second patient, a child of consanguineous parents from Tunisia, is homozygous for the unknown nonsense mutation W263X. This is the first report of a mutation, C53S, that affects the region of the keratinocyte transglutaminase that is essential for anchorage of the enzyme to the plasma membrane. A novel, rapid in situ transglutaminase activity assay revealed the absence of keratinocyte transglutaminase activity in both patients. The mutations described are hence causative for the ichthyosis phenotype. Received: 27 October 1997 / Accepted: 24 November 1997     

Cinnamic,myristic and fumaric acids in tobacco root exudates induce the infection of plants by Ralstonia solanacearum

Aim

The secretion of allelochemicals from plant roots plays a key role in soil sickness and soil-borne disease. The goal of this study was to investigate the role of allelopathic chemicals in Ralstonia solanacearum-infected tobacco roots.

Methods

The organic acids investigated in the present study are major components of tobacco root exudates. Through a swarming assay, we assessed the chemotaxis and colonization of R. solanacearum in response to organic acids.

Results

Fumaric acid was detected, and the results showed that this acid could serve as a semiochemical for attracting R. solanacearum and inducing the formation of biofilms of this species. The results also revealed that cinnamic and myristic acids play significant roles on swarming motility and chemotaxis. In addition, cinnamic, myristic and fumaric acids could enhance the expression of chemotaxis- and motility-related genes in R. solanacearum cultured in minimal medium. Furthermore, these three acids promote R. solanacearum colonization and accelerate disease progression in tobacco.

Conclusion

Cinnamic, myristic and fumaric acids could serve as semiochemical attractants to induce the colonization and infection of R. solanacearum. The results of the present study enhance our understanding of the ecological effects of plant root exudates in plant-microbe interactions and help to reveal the relationship between tobacco bacterial wilt and the autotoxins and allelochemicals that accumulate from root exudates.

     

Placental transport of free palmitic and linoleic acids in the guinea pig

Radioisotopic tracers were used to measure the unidirectional transfer rates of free fatty acids across the placenta of fed and fasted pregnant guinea pigs. Free (14)C-labeled palmitic and linoleic acids (in serum) were injected simultaneously into a jugular vein of an anesthetized pregnant guinea pig. Serial samples of maternal blood were collected from a carotid artery; fetal blood was collected from the umbilical vein of an exposed fetus. Analysis of maternal and fetal plasma revealed that: (a) the half-lives of free palmitic and linoleic acid in maternal plasma are approximately 1.3 min and 0.7 min, both in fed animals with low plasma concentrations of these acids and in fasted animals with high concentrations; (b) free linoleic and palmitic acids cross the placenta from maternal to fetal plasma in a ratio of approximately 2.0, a value which appears not to change as the transfer rates of these acids from maternal to fetal plasma are increased by fasting the mother. It is suggested that the ratio in which free linoleic and palmitic acids cross the placenta from maternal to fetal plasma is determined by the ratio of the unbound free linoleic and palmitic acid concentrations in maternal plasma. A comparison of several species indicates that a much greater proportion of fetal fatty acids comes from the mother in the guinea pig and rabbit than in the rat, the sheep, or man.     

Acylation of carcinogenic hydroxamic acids by carbamoyl phosphate to form reactive esters

Two methylated derivatives of arginine were isolated from the encephalitogenic protein of myelin from the central nervous system. Evidence is presented for the proposed structures, omega-NN'-dimethylarginine and omega-N-monomethylarginine. In the encephalitogenic protein from human brain the proportion 1:6:10 for arginine:monomethylarginine:dimethylarginine residues was found to occur at position 107. Possible roles for the methylated arginine in conformational changes and altered ion-exchange behaviour are discussed.     

Plasma membrane transglutaminase and cytosolic transglutaminase form distinct envelope-like structures in transformed human keratinocytes

Cross-linked envelope formation in the transformed human keratinocyte line SV-K14 requires treatment of the cells with a Ca2+ ionophore. Depending on the culture conditions, different extracellular Ca2+ concentrations are necessary to trigger the process which is catalyzed by the enzyme transglutaminase. Confluent cells grown in the presence of serum express only the cytosoluble form of the enzyme and need 5 mM Ca2+ for optimum protein cross-linking, whereas serum-starved cells which additionally contain the plasma membrane associated form of the enzyme require only 1 mM Ca2+. The envelope-like structures thus synthesized are morphologically and biochemically distinct.     

Alterations in membrane fluidity during keratinocyte differentiation measured by fluorescence polarization

Summary The epidermis shows a distinctive pattern of differentiation wherein keratinocytes proliferate in the basal cell layer and mature into spinous and granular cells. Using a discontinuous density-gradient centrifugation method, guinea-pig keratinocytes were separated into high (HDF), intermediate (IDF), and low (LDF) density fractions. Morphological and flow cytometrical observations demonstrated that HDF, IDF, and LDF were basal, spinous, and granular cell-rich fractions, respectively. Membrane fluidity of the fractionated keratinocytes was measured by diphenylhexatriene fluorescence polarization. Polarization (p)-value of keratinocytes was negatively correlated with temperature. At each temperature, HDF cells showed a lower p-value than IDF or HDF cells except at 40° C. Since a low p-value indicates a high degree of Brownian motion, membrane fluidity is higher in basal cells and lower in spinous and granular cells. Our results indicate that membrane fluidity of guinea-pig keratinocytes decreases during their maturation.     

Metabolism of palmitic and docosahexaenoic acids in Reuber H35 hepatoma cells

In this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (palmitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that palmitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.