Non-volatile components of the essential oil secretory cavities of Eucalyptus leaves: Discovery of two glucose monoterpene esters, cuniloside B and froggattiside A

The essential oils extracted from the embedded foliar secretory cavities of many Eucalyptus species are of economic value as pharmaceuticals and fragrance additives. Recent studies have indicated that Eucalyptus secretory cavities may not be exclusively involved in the biosynthesis and storage of essential oils. Therefore, we selected three species upon which to perform an examination of the contents of foliar secretory cavities: Eucalyptus froggattii, E. polybractea and E. globulus. This paper describes the isolation and structural characterization of two non-volatile glucose monoterpene esters, which we have named cuniloside B and froggattiside A, from within the secretory cavities of these species, and shows the presence of these compounds in solvent extracts of the leaves from two other species of Eucalyptus. Both compounds were found in high proportions relative to the essential oils extracted from the leaves. We propose that many other carbohydrate monoterpene esters previously isolated from bulk leaf extracts of various Eucalyptus species may also be localized within the non-volatile fraction of foliar secretory cavities.     

Structure and development of the secretory cavities of Myrtus communis leaves

The structure and development of Myrtus communis L. secretory cavities has been studied in young and expanded leaves, using light and scanning electron microscope. Secretory cavities are continuously formed during leaf development, but in mature leaves the rhythm of their appearance shows steep decrease. Each secretory cavity is developed from a single epidermal cell, which undergoes a periclinal division followed by anticlinal and several oblique cell divisions. The lumen of the secretory cavity is initiated by cell wall separation, i.e., schizogenously. The secretory cells line the cavity, where the secreted material is collected. Secretory cavities are covered by modified epidermal cells, which do not seem to form any special aperture. Essential oils seem to be discharged after mechanical treatment of the leaf.     

Ellagitannins of the casuarinaceae,stachyuraceae and myrtaceae

Eight ellagitannins and related polyphenols, found in Casuarina stricta and Stachyurus praecox, were detected in the leaf of Psidium species. Five of them and a new polyphenol, named isostrictinin, were isolated from Psidium guajava. Most of these compounds were detected in several species of Myrtaceae, and 2, 3-hexahydroxydiphenoylglucose and 4, 6-hexahydroxydiphenoylglucose were found in some.     

Localization of the glucose phosphotransferase to a cytoplasmically accessible site on intracellular membranes

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in rat liver is a glycoprotein of 62 kDa. This acceptor was labeled in liver homogenates through incubation with the 35S-labeled phosphorothioate analogue of UDP-Glc, and its distribution following differential centrifugation was compared to that of the glycoproteins labeled by CMP-[3H]N-acetylneuraminic acid. Whereas 94% of the 3H-labeled macromolecules fractionated to the microsomal pellet, 85% of the 35S-labeled 62-kDa glycoprotein was found in the high-speed supernatant. The distribution of the Glc-phosphotransferase was also examined following differential centrifugation, and the bulk of the activity was found in the 100,000 x g pellet. In contrast to results obtained with the lumenal microsomal markers 4 beta-galactosyltransferase and mannose-6-phosphatase, however, optimal activity of the Glc-phosphotransferase was not dependent on the disruption of microsomal vesicles by detergent. In addition, Glc-phosphotransferase was degraded by exogenous proteases in the absence of detergent, whereas the lumenal markers were not. We conclude, therefore, that the 62-kDa acceptor glycoprotein is cytoplasmic and is glycosylated by the Glc-phosphotransferase at a site accessible to the cytoplasm. This may prove to be a model for the topography of glycosylation of other cytoplasmic glycoproteins as well.     

Localization of epididymal secretory proteins on rat spermatozoa

Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.     

Preparation of various glucose esters via lipase-catalyzed hydrolysis of glucose pentaacetate

beta-D(+)-Glucose pentaacetate was hydrolyzed both chemically and enzymatically. In contrast to the alkaline hydrolysis, esterase-catalyzed deacetylations afforded significant accumulation of intermediate glucose esters at different degrees of substrate conversion. Aspergillus niger lipase, the most suitable of the four enzymes tested, was used for preparative hydrolysis of glucose pentaacetate. As a result, gram quantities of pure glucose-2,3,4,6-tetraacetate, glucose triacetate (a mixture of two positional isomers, 2,4,6- and 3,4,6-), and glucose-4,6-diacetate were prepared.     

Anatomical and chemical analyses of leaf secretory cavities of Rustia formosa (Rubiaceae)

Foliar secretory cavities, commonly called leaf pellucid glands, have been reported in many families of vascular plants. In the Rubiaceae, these structures have only been found in the sister genera Rustia and Tresanthera, which are also anomalous within the family because they have poricidal anthers, and in the distantly related Heterophyllaea. General leaf anatomy, with particular attention to secretory cavities, as well as the chemical analysis of the secreted substances of Rustia formosa, is presented here for the first time. The secretory structures have been found in the lamina between the palisade and spongy parenchymas and in the cortical region of the petiole. The chemical analysis showed that the essential oil secreted is a complex mixture of at least 75 components, mostly of sesquiterpenoid composition. Illustrations of the leaf anatomy, details of the secretory structures of Rustia formosa, a gas chromatogram, and a table of the principal components of the leaf essential oil are included.     

A flavanone and a dihydrodibenzoxepin from Bauhinia variegata

Phytochemical analysis of the root bark of Bauhinia variegata Linn yielded a new flavanone, (2S)-5,7-dimethoxy-3',4'-methylenedioxyflavanone (1) and a new dihydrodibenzoxepin, 5,6-dihydro-1,7-dihydroxy-3,4-dimethoxy-2-methyldibenz [b,f]oxepin (2) together with three known flavonoids (3-5). The structures of the new compounds were determined on the basis of spectral studies.     

Spontaneous and enzymic rearrangement of naringenin chalcone to flavanone

The isomerisation of 2′,4,4′,6′-tetrahydroxychalcone by the enzyme chalcone isomerase is difficult to assay accurately in view of the spontaneous cyclisation of this chalcone at the alkaline pH optimum of the enzyme. We report here that self-cyclisation of naringenin chalcone is dramatically reduced at pH-values ? 6.5 and in the presence of high concentrations of serum albumin (5–10 mg ml ^?1). We have critically evaluated existing assay procedures of chalcone isomerase, utilizing the effects of a monospecific anti-(chalcone isomerase) serum to distinguish between spontaneous and enzymic cyclisation of chalcone. We conclude that the modifications listed above considerably facilitate the measurement of chalcone isomerase kinetics.     

Dietary therapy and insulin secretory response to glucose in adult-onset non-obese diabetic subjects.

The effect of a 4-week diet regulation on non-obese, adul-onset diabetics was studied. The diet, which was prescribed for them, was composed of 60% carbohydrate, 15-20% protein and 20-25% fat. The total caloric intake was restricted to 30, 35 and 40 Cal/kg ideal body weight depending on their physical activity. In the group whose calculated diet showed over 10% reduction in total caloric intake and carbohydrate intake, fasting glucose was decreased and glucose tolerance was improved significantly after the 4-week dietary therapy. Insulin response to oral glucose loading was improved, particularly in the later stage of oral glucose tolerance test. As a result, insulin area, i. e. the total area under the insulin curve was increased to almost two times. The sensitivity to insulin did not show any significant changes after diet regulation. The present data indicate that the therapeutic effect of the diet restriction should be at least in part ascribed to the increased secretion of insulin. In the treatment of diabetics, a restricted diet is essential and beneficial from the point of view that it could improve the pancreatic beta-cell function.     

Localization of glycoproteins in insulin secretory granules by ultrastructural autoradiography

To determine whether or not the secretory granules of insulin-secreting cells contained glycoproteins, isolated rat pancreatic islets were incubated for 2 and 4 hr in a medium containing L-[3H]-fucose. Quantitative analysis of high-resolution electron microscopic autoradiographs of the insulin-secreting beta cells demonstrated that glycoproteins with fucose residues are contained within the insulin secretory granule.     

Localization of calcium in the secretory cells of the mammary glands in response to oxytocin

Distribution of Ca2+ ions, precipitated by means of pyroantimonate potassium, has been investigated electron microscopically in secretory cells of the mammary gland of lactating white mice. In the glandular cells, that are at the state of inhibition of secretory activity, the cytochemical reaction product is localized on the internal side of the basal, lateral and apical parts of the plasmolemma, in mitochondrial matrix, in cisterns and in the Golgi complex vesicles, in the nuclear areas, occupied by euchromatin. Oxytocin effect produces a certain complex of ultrastructural changes in the cell accompanied by redistribution of Ca2+ ions. Amount of precipitate in mitochondria decreases. It is revealed in the lumen of dilated canals of the granular endoplasmic reticulum, in the zone of decondensated nuclear chromatin, in the Golgi complex vesicles. The vesicles become larger and fuse with each other. The changes mentioned demonstrate increased synthetic and transport processes, occurring in the glandular epithelium of the mammary gland after oxytocin effect.     

Localization of plasma membrane and secretory calcium pumps in the mammary gland

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.     

Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner

Recent evidence has shown that activation of lipid-sensitive protein kinase C (PKC) isoforms leads to skeletal muscle insulin resistance. However, earlier studies demonstrated that phorbol esters increase glucose transport in skeletal muscle. The purpose of the present study was to try to resolve this discrepancy. Treatment with the phorbol ester 12-deoxyphorbol-13-phenylacetate 20-acetate (dPPA) led to an approximately 3.5-fold increase in glucose transport in isolated fast-twitch epitrochlearis and flexor digitorum brevis muscles. Phorbol ester treatment was additive to a maximally effective concentration of insulin in fast-twitch skeletal muscles. Treatment with dPPA did not affect insulin signaling in the epitrochlearis. In contrast, phorbol esters had no effect on basal glucose transport and inhibited maximally insulin-stimulated glucose transport approximately 50% in isolated slow-twitch soleus muscle. Furthermore, dPPA treatment inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the threonine and serine phosphorylation of PKB by approximately 50% in the soleus. dPPA treatment also caused serine phosphorylation of IRS-1 in the slow-twitch soleus muscle. In conclusion, our results show that phorbol esters stimulate glucose transport in fast-twitch skeletal muscles and inhibit insulin signaling in slow-twitch soleus muscle of rats. These findings suggest that mechanisms other than PKC activation mediate lipotoxicity-induced whole body insulin resistance.     

Localization and secretory pathways of a 58K-like protein in multi-vesicular bodies in callus of Arabidopsis thaliana

Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.     

Remains of secretory cavities in pinnules of Stephanian pteridosperms from Blanzy-Montceau (Central France): a comparative study

Remains of secretory cavities, so-called resin or secretion bodies, in pinnules of Stephanian pteridosperms from the Blanzy-Montceau Basin (Central France) are examined from cuticles of four species: Dicksonites leptophylla, D. pluckenetii, Pseudomanopteris paleaui , and P. ribeyronii . The distribution of the secretion bodies in pinnules may be (1) simple and in consistent patterns, (2) random, but topographically unequal, or (3) completely random. Differences in the morphology of the bodies may be related to different modes of development of the secretory cavities, i.e. schizogenous or (schizo-) lysigenous development. The value of using these features in fossil plant taxonomy is discussed. Finally, some comments on possible ecological functions of the secretion of lipophilic substances in pteridosperm pinnules are suggested. The secretion of lipophilic substances is abundant among taxa with a vine-like life-form, and this may be related to special demands on protection of climbing or scrambling plants.     

Lipase-catalysed synthesis of glucose fatty acid esters in tert-butanol

Synthesis of 6-O-acylate--d-glycopyranose from underivatised substrates in anhydrous tert-butanol was achieved using immobilised lipases from Candida antarctica and Mucor miehei. Except for acetic acid, the initial reaction rates with the C. antarctica lipase were independent of acyl donor chain lengths and in a range of 3.9±0.4 mol glucose converted min–1 g enzyme preparation. The catalytic activity of the M. miehei lipase increased with increasing acyl donor chain length with a maximum for stearic acid of 0.45 mol min–1 g. Using maltose as substrate, the catalytic activity decreased by a factor of 48 and 20 with the lipase from C. antarctica and M. miehei, respectively, while with maltotriose no reaction was observed.     

Derepression of high-affinity glucose uptake requires a functional secretory system in Saccharomyces cerevisiae.

The expression of high-affinity glucose uptake in Saccharomyces cerevisiae strains carrying conditional mutations conferring a block of secretion and cell surface growth (sec) revealed a requirement for a functional secretory pathway for derepression of carrier activity. Thus, in strains carrying the sec1-1, sec4-2, sec7-1, sec14-3, or sec17-1 mutation, no high-affinity carrier activity was expressed after a shift to derepressing glucose concentrations at the nonpermissive temperature. In the case of sec18-1, however, derepression of carrier activity did occur at both the permissive and nonpermissive temperature, but not to the same extent as found in the wild-type strain, suggesting that SEC18 function may not be essential for expression of carrier activity. In sec1-1, accumulation of high-affinity carrier activity (or a component thereof) in presecretory vesicles during incubation at the nonpermissive temperature was demonstrated. The presence of a high glucose concentration in the medium did not affect transfer of that accumulated carrier function to the cell surface. Carrier function did not accumulate in strains carrying the other sec mutations. Analysis of the stability of high-affinity carrier activity at 37 degrees C demonstrated rapid and unexpected loss of carrier activity not affected by the presence of glucose in the medium. Thus, blockage of cell surface growth seems to affect turnover rates of hexose carrier activities.     

Observations on the development of the foliar secretory cavities of Porophyllum lanceolatum (Asteraceae)

The lipophilic secretory cavities observed in the leaf of Porophyllum lanceolatum (Asteraceae) are scattered throughout the lamina and around its crenate margins. In the young leaf the cavities are initiated, and their development completed, while the surrounding tissues are still at early stages of differentiation. The cavity lumen has a lysigenous origin. Cell lysis, expansion of the developing leaf and, probably, the pressure exerted by the accumulation of secretory products, are believed to account for the gradual enlargement of the lumen. Concomitantly with ctll disintegration, which occurs throughout development, divisions take place in all cells of the gland. A mature cavity has a multilayered epithelium. Histochemical tests for RNA, proteins, phenolics and pectic polysaccharides revealed intense staining of the content of the epithelial cells in the early stages of cavity development, and a decrease in staining towards its maturity. Staining for lipids is intense in all developmental stages. Tests on the material observed in the lumen of mature cavities, show positive results for lipids, pectic polysaccharides and phenolics.