Structural and histochemical changes during seed development of Brassica macrocarpa Guss.

During seed formation of Brassica macrocarpa the development of the embryo precedes that of the integuments; structural changes and histochemical changes are associated. Esterases, acid phosphatases, phenols and starch follow a sigmoid pattern, increasing during embryogenesis and decreasing during seed maturation. In the mature seed, esterase activity is localized in the embryo and in the cells of the mucilaginous, aleuronic and hyaline layers. Acid phosphatases are present in the mucilaginous cells, mainly in the column, the cell walls delimiting intercellular spaces of the cortical cylinder and the adhesion areas of the cotyledons. Phenols are scanty in the root apex, mucilaginous cells and the palisade layer, and abundant in the pigmented layer. Starch is absent in ripe seeds which have lipid and protein reserves. The major classes of storage proteins have molecular weights of 21, 22, 27 and 30 KD and accumulate in the late stages prior to complete drying. Esterases and acid phosphatases in mucilaginous cells of the seed integument suggest that these enzymes are involved in hydrolytic processes occurring prior to germination and that mucilages have a metabolic function in seed-soil interactions.     

Discovery of new G protein-coupled receptors for lipid mediators

Successful sequencing of the human genome has opened a new era in the life sciences and has greatly accelerated biomedical research. Among various research endeavors benefiting from established genomic information, one of the most fruitful areas is the research on orphan G protein-coupled receptors (GPCRs). Many intercellular mediators, including peptides, lipids, and other small molecules, have found their GPCRs in the plasma membrane, e.g., relaxin and tyramine. In the past 14 months, more than one dozen papers have been published reporting the finding of intercellular lipid mediators acting on rhodopsin family GPCRs. This review focuses primarily on intercellular lipid mediators and their recently discovered GPCRs.     

Novel method to observe subtle structural modulation of stratum corneum on applying chemical agents

In the development of functional chemicals such as percutaneous penetration enhancers and cosmetics, the structural evidence at the molecular level in stratum corneum (SC) is highly desirable. We developed a method to observe a minute structural change of intercellular lipid matrix and corneocytes on applying the chemicals to the SC using synchrotron X-ray diffraction technique. The performance of the present method was demonstrated by applying typical chemicals, chloroform/methanol mixture, hydrophilic ethanol and hydrophobic d-limonene. From the small- and wide-angle X-ray diffraction we obtained the following results: on applying chloroform/methanol mixture the intercellular lipids were extracted markedly, on applying ethanol the intercellular lipid structure was slightly disrupted, ethanol molecules were taken into the corneocytes and in addition the pools of ethanol seem to be formed in the hydrophilic region of the intercellular lipid matrix in the SC, and on applying d-limonene the repeat distance of the long lamellar structure increased by incorporating d-limonene molecules, the intercellular lipid structure was slightly disrupted, and the pools of d-limonene were formed in the hydrophobic region of the intercellular lipid matrix in the SC.     

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [³H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.     

Effect of a sake concentrate on the epidermis of aged mice and confirmation of ethyl alpha-D-glucoside as its active component

Generations of Japanese have appreciated the positive effects that sake can have on skin conditions, and studies have shown that concentrated sake suppressed the epidermal barrier disruption caused by ultraviolet B (UVB) irradiation. We investigated the effect of a topical application of a sake concentrate on the murine epidermis and found that the intercellular lipid content in an aged epidermis was significantly increased. Furthermore, the topical application of ethyl alpha-D-glucoside (alpha-EG), a component of sake, brought about a similar improvement in the levels of intercellular lipids. Following on from this, we confirmed that alpha-EG also significantly increased the content of loricrin protein, an indicator of successful corneocyte differentiation, while reducing the number of corneocyte layers in the aged stratum corneum. These results confirmed alpha-EG as the primary active component of the sake concentrate that had a positive effect on the epidermis. alpha-EG increased the intercellular lipid content, accelerated the differentiation of corneocytes, and reduced the thickness, thus improving the functions of the stratum corneum.     

Studies on seeds. II. Origin and degradation of lipid vesicles in pea and bean cotyledons

At least two kinds of lipid vesicles are present in pea and bean cotyledons which can be recognized at seed maturity on the basis of whether they do or do not interassociate into lipid vesicle sheets. Those that do interassociate into sheets are also characterized by (a) their association with plastids or plasma membranes during dormancy, and (b) the unique transformation into flattened saccules that they undergo during the first few days of seed germination. These interassociated (or composite) lipid vesicles have been found in only a few seeds and may be restricted to certain classes of plants and/or certain states of cellular development. Lipid vesicle-to-saccule transformation is predominantly confined to the germinating seed. However, some lipid vesicle-derived saccules are already present in some cells even before the seed reaches maturity. These partially transformed vesicles and saccules remain unchanged over dormancy, and then resume their transformation when the seed is germinated. This suggests that some stages of seed germination are already underway before the seed reaches maturity and are only resumed at seed germination. The lipid vesicles that do not interassociate into sheets (i.e., the simple lipid vesicles) are present in all tissues at all states of cellular development. These vesicles do not undergo any conspicuous structural changes during development.     

Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide

Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO₄-fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate.     

Drought stress during soybean seed filling affects storage compounds through regulation of lipid and protein metabolism

Soybean seeds have high lipid and protein contents. Adverse environmental conditions restrict seed yield and quality. We examined the changes in storage compounds caused by drought stress from R5 stage (beginning seed growth stage). Under drought stress, contents of lipid in seed were remarkably low compared to control at 24 and 29 days after treatment. Protein contents in seed were immediately decreased after water deficit treatment. On the other hand, soluble sugar contents in seed were increased by drought stress. Drought stress decreased the expression of genes involved in lipid biosynthesis (PK, BCCP2, and KAS1) and increased the genes expression involved in lipid degradation (ACX2, MS, and PEPCK). These results suggest that the increasing of sugar content in seed under drought stress was complemented by degradation of lipids. The expressions of genes encoding storage protein (Gy4 and β-conglycinin) were also decreased by drought stress. This study showed how drought stress during seed filling affects seed quality, especially lipid and protein contents, that may facilitate further research on seed storage compounds metabolism under environmental stresses.     

The modulation of gap-junctional intercellular communication by lipid rafts

Lipid rafts are specific microdomains of plasma membrane which are enriched in cholesterol and sphingolipids. These domains seem to favour the interactions of particular proteins and the regulation of signalling pathways in the cells. Recent data have shown that among the proteins, which are preferentially localized in lipid rafts, are connexins that are the structural proteins of gap junctions. Since gap junctional intercellular communication is involved in various cellular processes and pathologies such as cancer, we were interested to review the various observations concerning this specific localization of connexins in lipid rafts and its consequences on gap junctional intercellular communication capacity. In particular, we will focus our discussion on the role of the lipid raft-connexin connection in cancer progression. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Number of aberrations per cell as a parameter of chromosome instability. 2. Comparative analysis of the factors of different nature

The average number of aberrations per aberrant cell was concluded to carry out information on chromosome instability peculiarities induced by different mutagens as it was shown in our previous work. The purpose of the current study was to present comparative analysis of intercellular distribution of number of aberrations and their theoretical approximations. Distribution of numbers of aberrations per cell in Allium cepa L. and Allium fistulosum L. root tip cells induced by different mutagenic factors (gamma-irradiation, thiotepa, formaldehyde and seed aging) have been studied. The results were approximated to theoretical Poisson, geometric and negative binomial distributions. The intercellular distribution of aberrations did not correspond to any of the used theoretical distributions when A. cepa seeds were gamma-irradiated. There was some, but not regular, accordance with theoretical distributions when chemical mutagens thiotepa in A. cepa and formaldehyde in A. fistulosum and seed aging in both species were evaluated. During seed aging frequency of aberrant cells increased more quickly in A. fistulosum in comparison with A. cepa.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

Molecular composition and surface properties of storage lipid particles in wax bean (Phaseolus vulgaris)

Lipid particles have been isolated from seeds of wax bean (Phaseolus vulgaris), a species in which starch and protein rather than lipid are the major seed storage reserves. These lipid particles resemble oil bodies present in oil-rich seeds in that > 90% of their lipid is triacylglycerol. Moreover, this triacylglycerol is rapidly metabolized during seed germination indicating that it is a storage reserve. The phospholipid surfaces of oil bodies are known to be completely coated with oleosin which prevents their coalescence, particularly during desiccation of the developing seed. This would appear to be necessary since lipid is the major storage reserve in oil seeds, and there are very few alternate types of storage particles in the cytoplasm of oil seed endosperm to provide a buffer against coalescence of oil bodies by isolating them from one another. The present study indicates that the surfaces of lipid particles from wax bean are not completely coated with oleosin and feature regions of naked phospholipid. This finding has been interpreted as reflecting the fact that lipid particles in wax been seeds are less prone to coalescence than oil bodies of oil-rich seeds. This arises because the individual lipid particles are interspersed in situ among highly abundant protein bodies and starch grains and hence less likely to come in contact with one another, even during desiccation of the developing seed.     

The epidermal permeability barrier. Comparison between in vivo and in vitro lipid structures

The epidermal permeability barrier is established by the lamellar contents of membrane-coating granules which are discharged into the intercellular space of the stratum granulosum and form continuous lipid layers in the stratum corneum. Artificial lipid systems, prepared with a composition similar to that found in stratum granulosum and stratum corneum, were able to form a lamellar phase. These systems show dense line thickness and center-to-center spacing comparable to those found in membrane-coating granules and intercellular layers. The significance of lipid composition in relation to barrier function is discussed and a model showing the molecular arrangement of the lipid structures in the epidermal barrier is proposed.     

Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study

The lipid of the outermost layer of the skin is confined largely to the extracellular spaces surrounding the corneocytes of the stratum corneum where it forms a multilamellar adhesive matrix to act as the major permeability barrier of the skin. Knowledge of the molecular architecture of these intercellular domains is important for understanding various skin pathologies and their treatment, percutaneous drug delivery, and the cosmetic maintenance of the skin. We have surveyed by X-ray diffraction the structure of the intercellular domains and the extracted lipids of murine stratum corneum (SC) at 25, 45, and 70 degrees C which are temperatures in the vicinity of known thermal phase transitions [Rehfeld, S. J., & Elias, P. M. (1982) J. Invest. Dermatol. 79, 1-3]. The intercellular domains produce lamellar diffraction patterns with a Bragg spacing of 131 +/- 2 A. Lipid extracted from the SC and dispersed in excess water does not produce a simple lamellar diffraction pattern at any temperature studied, however. This and other facts suggest that another component, probably a protein, must be present to control the architecture of the intercellular lipid domains. We have also obtained diffraction patterns attributable to the protein envelopes of the corneocytes. The patterns suggest a beta-pleated sheet organizational scheme. No diffraction patterns were observed that could be attributed to keratin.     

The nanoscopic molecular pathway through human skin

BackgroundKnowledge regarding the barrier properties of human skin is important for understanding skin pathology, developing of transdermal drug delivery systems and computational skin absorption models; however, the molecular pathways through human skin remains to be fully investigated on a nanoscopic level. In particular the nanoscopic pathway of molecules passing the intercellular lipid bilayers separating the corneocytes in the stratum corneum (SC) is not fully elucidated.MethodsUsing stimulated emission depletion microscopy (STED) and Förster resonance energy transfer (FRET) the molecular pathways through the SC, the main barrier of the skin, are determined for lipophilic and water-soluble molecules at a nanoscopic resolution.ResultsUsing STED and confocal microscopy, water-soluble dyes, were observed to be present in both the corneocytes and in the intercellular lipid matrix, whereas the lipophilic dyes were predominately in the intercellular lipid bilayers. FRET was observed in the SC between the lipophilic and water-soluble dyes, the existence of a minimum possible distance between acceptor and donor molecules of 4.0 ± 0.1 nm was found.ConclusionsThe results indicate that lipophilic molecules penetrate the stratum corneum via the intercellular lipids bilayers separating the corneocytes in the SC, while the more water-soluble molecules penetrate the stratum corneum via the transcellular route through the corneocytes and intercellular lipid bilayers via the polar head groups of lipid molecules in the bilayers.General significanceKnowledge of the nanoscopic molecular pathways through human skin will help understand the skin barrier function and will be of use for computational skin absorption models and transdermal drug delivery strategies.     

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.     

Protein modification by Amadori and Maillard reactions during seed storage: roles of sugar hydrolysis and lipid peroxidation

The non-enzymatic modifications of proteins through Amadori and Maillard reactions play an important role in the loss of seed viability during storage. In the present study, the contribution of sugar hydrolysis and lipid peroxidation to Amadori and Maillard reactions, and to seed deterioration was investigated in mung-bean (Vigna radiata Wilczek). The contents of glucose and lipid peroxidation products in seed axes increased significantly during storage. The accumulation of Amadori products in seed axes was correlated to the lipid peroxidation, whereas the accumulation of Maillard products was closely correlated to sugar hydrolysis. The rate of accumulation of Maillard products was not well correlated to the content of Amadori products in both seed axes and protein/glucose model system, reflecting the complex nature of Amadori and Maillard reactions. The content of Amadori products in seed axes increased during the early stages of seed ageing, whereas the content of Maillard products increased steadily during the entire period of storage. The accumulation of Maillard products in seed axes was associated with the decline of seed vigour. These data suggest that, during seed ageing, sugar hydrolysis and lipid peroxidation are coupled with non-enzymatic protein modification through Amadori and Maillard reactions.     

Changes in lipid status and glass properties in cotyledons of developing sunflower seeds

Biochemical events involved in the acquisition of germinability and storability during orthodox seed development are well documented; however, the roles played by the physical organization of lipids and water are poorly characterized. The aim of this work was to determine, using a thermodynamic approach, whether changes in thermal properties of lipid reserves, and intracellular glasses might play a role in sunflower (Helianthus annuus L.) seed development. Triacyglycerols (TAGs) accumulated in cotyledons until the end of seed filling, which occurred 42 days after anthesis (DAA). Further seed development, leading to mature seed at 58 DAA, was mainly associated with an enlargement of lipid bodies without significant changes either in the lipid content or in their composition. When cooled to -100 degrees C, lipid reserves from cotyledons of mature seeds displayed alpha and beta' polymorphic crystalline structures; however, the ability to form alpha crystals, which was an indicator of lipid purity, progressively appeared during seed development. Characteristics of lipid melting confirmed that seed maturation drying was associated with changes in TAG physical organization. Cotyledon development was associated with an increase in the temperature of glass to rubber transition (Tg), thus suggesting a decrease in molecular mobility during maturation drying. This phenomenon was concomitant with an increase in raffinose content. Our results demonstrate that physical characteristics of lipid reserves and glasses of sunflower cotyledons are developmentally regulated and might play a role in acquisition of seed germinability and storability.     

Mycorrhizal influence on protein and lipid of durum wheat grown at different soil phosphorus levels

 Root colonization by arbuscular mycorrhizal fungi (AMF) may affect protein and lipid composition of plants by altering P nutrition or by eliciting other metabolic responses in the host plant. This study was conducted to determine the effects of an AMF and soil P on seed protein and lipid contents and yield of two genotypes of durum wheat (Triticum durum L.). Plants were grown in a greenhouse using soil: sand mixes with different levels of P, and with or without the AMF Glomus mosseae [(Nicol. and Gerd.) Gerd. and Trappe]. Percentage AMF root colonization decreased as P added to soil increased. The wheat genotype CR057 had higher AMF root colonization but lower seed P and protein concentrations than CR006. Without added soil P, protein concentration was significantly lower and lipid concentration and seed dry weight higher in arbuscular mycorrhizal (AM) than in nonAM plants. Seed lipid and protein contents were highly correlated with P content of plants. In nonAM plants, seed lipid and protein contents were low with no added soil P and did not differ with added soil P. Seed protein/lipid (Pro/L) concentration ratios of AM plants were higher than those of nonAM plants only when no P was added to the soil. The data indicate different patterns of seed P accumulation and different relationships between seed P and protein and lipid in AM and nonAM plants. Thus, both the presence and degree of AMF root colonization affected seed lipid metabolism in these durum wheat genotypes. Accepted: 18 May 1999