New arrangement of proteins and lipids in the stratum corneum cornified envelope.

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.

Focused ion beam/scanning electron microscopy characterization of cell behavior on polymer micro-/nanopatterned substrates: a study of cell-substrate interactions.

Topographic micro and nanostructures can play an interesting role in cell behaviour when cells are cultured on these kinds of patterned substrates. It is especially relevant to investigate the influence of the nanometric dimensions topographic features on cell morphology, proliferation, migration and differentiation. To this end, some of the most recent fabrication technologies, developed for the microelectronics industry, can be used to produce well-defined micro and nanopatterns on biocompatible polymer substrates. In this work, osteoblast-like cells are grown on poly(methyl methacrylate) substrates patterned by nanoimprint lithography techniques. Examination of the cell-substrate interface can reveal important details about the cell morphology and the distribution of the focal contacts on the substrate surface. For this purpose, a combination of focused ion beam milling and scanning electron microscopy techniques has been used to image the cell-substrate interface. This technique, if applied to samples prepared by freeze-drying methods, allows high-resolution imaging of cross-sections through the cell and the substrate, where the interactions between the nanopatterned substrate, the cell and the extracellular matrix, which are normally hidden by the bulk of the cell, can be studied.

Effect of bicellar systems on skin properties.

Bicelles are discoidal aggregates formed by a flat dimyristoyl-glycero-phosphocholine (DMPC) bilayer, stabilized by a rim of dihexanoyl-glycero-phosphocholine (DHPC) in water. Given the structure, composition and the dimensions of these aggregates around 10-50 nm diameter, their use for topical applications is a promising strategy. This work evaluates the effect of DMPC/DHPC bicelles with molar ratio (2/1) on intact skin. Biophysical properties of the skin, such as transepidermal water loss (TEWL), elasticity, skin capacitance and irritation were measured in healthy skin in vivo. To study the effect of the bicellar systems on the microstructure of the stratum corneum (SC) in vitro, pieces of native tissue were treated with the aforementioned bicellar system and evaluated by freeze substitution applied to transmission electron microscopy (FSTEM). Our results show that bicelles increase the TEWL, the skin elastic parameters and, decrease skin hydration without promoting local signs of irritation and without affecting the SC lipid microstructure. Thus, a permeabilizing effect of bicelles on the skin takes place possibly due to the changes in the phase behaviour of the SC lipids by effect of phospholipids from bicelles.

Self-assembly of the amphipathic helix (VHLPPP)8. A mechanism for zein protein body formation.

gamma-Zein, a maize storage protein with an N-terminal proline-rich repetitive domain (gamma-ZNPRD), is located at the periphery of protein bodies. This domain appears to be indispensable for the aggregation of the protein on the surface of the organelle. The peptide (VHLPPP)8, spanning the gamma-ZNPRD, adopts a polyproline II (PPII) conformation that gives an amphipathic helix different from the alpha-helix. We used atomic force microscopy to study the surface organisation of the octamer, and transmission electron microscopy to visualise aggregates of the peptide in aqueous solution. We consider two self-assembly patterns that take account of the observed features. The micellar one fits best with the experimental results presented. Moreover, we found that this peptide has properties associated with surfactants, and form micelles in solution. This spontaneous amphipathic arrangement of the gamma-ZNPRD suggests a mechanism of gamma-zein deposition inside maize protein bodies.

Herpes simplex virus type 1-induced modifications in the distribution of nucleolar B-36 protein.

Nucleolar B-36 protein was localized ultrastructurally by immunocytochemistry with monoclonal antibody P2G3 and colloidal gold label in rabbit fibroblast cells before and during infection with herpes simplex virus (HSV) type 1. In non-infected cells, labeling was sparse and restricted to the fibrillar component of the nucleoli. During the infectious cycle, B-36 protein appeared to be somewhat more abundant within the morphologically altered fibrillar component of the nucleoli. In addition, the protein was also detected in some but not all virus-induced intranuclear dense bodies. These observations suggest the presence of functionally distinct dense bodies. The association of B-36 protein with both structures was not disrupted by a hypotonic shock and detergent treatment, which suggest that these sites do not represent areas of passive intranuclear diffusion. Inhibition of protein synthesis late in infection, viral DNA replication or RNA synthesis did not alter the distribution of B-36 protein. We suggest that this protein may play a role in the increased compaction of the ribonucleoprotein fibrils induced by HSV infection, perhaps in association with some of the virus-encoded proteins which also have been detected in the nucleoli.

Direct formation of mixed micelles in the solubilization of phospholipid liposomes by Triton X-100.

The vesicle to micelle transition which results in the interaction of the Triton X-100 surfactant with phosphatidylcholine vesicles was studied by means of dynamic light scattering (at different reading angles) and by freeze-fracture electron microscopy techniques. Vesicle solubilization was produced by the direct formation of mixed micelles without the formation of complex intermediate aggregates. Thus, vesicle to micelle transformation was mainly governed by the progressive formation of mixed micelles within the bilayer. A subsequent separation of these micelles from the liposome surface (vesicle perforation by the formation of surfactant-stabilized holes on the vesicle surface) led to a complete solubilization of liposomes.

A comparison of cryo- versus chemical fixation in the soil green algae Jaagiella.

Chemical fixation protocols provided unsatisfactory preserved material for ultrastructural studies on Jaagiella alpicola Vischer (Chlorophyta). Instead, several methods of rapid freeze fixation followed by freeze substitution were applied. For fast freeze fixation, two methods were tried: plunge immersion freezing in liquid propane using a home-made device, and projection against a copper block cooled by either liquid nitrogen or liquid helium. Each method furnished well fixed material. The quality of the fixed samples was quite similar whether propane or the cryoblock cooled with liquid nitrogen was used. Liquid helium, however, provided superior results. After fixation the samples were cryosubstituted, using acetone or methanol as organic solvent with a chemical fixative added. Acetone gave better results than methanol as a substitution solvent when high temperature embedding was performed. The best cryosubstitution for ultrastructural studies was that in which osmium tetroxide or a mixture of osmium tetroxide and urany acetate was used.

Bicellar systems as new delivery strategy for topical application of flufenamic acid.

In this work, bicellar systems, bilayered disc-shaped nanoaggregates formed in water by phospholipids, are proposed as a novel strategy for delivery of the anti-inflammatory flufenamic acid (FFA) to the skin. A comparative percutaneous penetration study of this drug in bicellar systems and other vehicles was conducted. The effects induced on the skin by the application of FFA in the different vehicles were analyzed by attenuated total reflectance-fourier transform infrared (ATR-FTIR). Additionally, using the microscopic technique freeze-substitution transmission electron microscopy (FSTEM) and X-ray scattering technique using synchrotron radiation (SAXS-SR), we studied the possible microstructural and organizational changes that were induced in the stratum corneum (SC) lipids and the collagen of the skin by the application of FFA bicellar systems. Bicellar systems exhibited a retarder effect on the percutaneous absorption of FFA with respect to the other vehicles without promoting disruption in the SC barrier function of the skin. Given that skin disruption is one of the main effects caused by inflammation, prevention of disruption and repair of the skin microstructure should be prioritized in anti-inflammatory formulations.

Bicellar systems as modifiers of skin lipid structure.

The characterization of different bicellar aggregates and the effects of these systems on the stratum corneum (SC) microstructure have been studied. Dynamic light scattering (DLS) and freeze fracture electron microscopy (FFEM) techniques showed that both of the systems studied, dimyristoyl-phosphatidylcholine/dihexanoyl-phosphocholine (DMPC/DHPC) and dipalmitoyl-phosphocholine (DPPC)/DHPC, were formed by small discoidal aggregates at room temperature (20°C). Treating skin with DMPC/DHPC bicelles does not affect the SC lipid microstructure, whereas bicellar systems formed by DPPC and DHPC can promote the formation of new structures in the SC lipid domains. This indicates the passage of lipids from bicelles through the SC layers and also a possible interaction of these lipids with the SC lipids. Given the absence of surfactant in the bicellar composition and the small size of these structures, the use of these smart nano-systems offers great advantages over other lipid systems for dermatological purposes. Bicelles could be promising applications as drug carriers through the skin. This contribution, based on the new biological use of bicelles, may be useful to scientists engaged in colloid science and offers a new tool for different applications in skin and cosmetic research.

Lipid nanostructures: self-assembly and effect on skin properties.

This work evaluates the relation between the composition and the self-assembly of some lipid aggregates with their effects on the skin. To this end, liposomes, bicelles and micelles formed by dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) were characterized by electron microscopy and dynamic light scattering techniques, and applied on the skin. The results revealed that nanostructures with similar assembly but different composition caused different effects on the skin parameters. In general, samples containing DMPC affected the barrier function to a greater extent than systems containing DPPC. Additionally, our results showed that samples with the same lipid composition but different assembly exerted different effects on the skin. Liposomes decreased or did not modify the transepidermal water loss (TEWL), while bicelles and micelles increased this parameter. Hydration of the skin diminished especially after the application of micellar and bicellar samples. In vitro experiments showed structures like vesicles inside cutaneous SC (stratum corneum) incubated with DPPC/DHPC bicelles. These structures were not detected in SC samples incubated with DMPC/DHPC bicelles probably due to the different thermotropic behavior of DMPC and DPPC at physiological temperatures. Results reported in this work should be considered in terms of design of more efficient and specific skin delivery systems.

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

We have analysed the incorporation of [(3)H]sucrose and [(3)H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [(3)H]sucrose or [(3)H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2-3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [(3)H]Sucrose uptake was twice that of [(3)H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [(3)H]sucrose and [(3)H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [(3)H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [(3)H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [(3)H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Fatty acyl moieties: improving Pro-rich peptide uptake inside HeLa cells.

In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro-rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro-rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.

Ultrastructural localization of glycoproteins in rabbit fibroblasts altered by Herpes simplex virus type 1 infection.

The periodic acid-thiocarbohydrazide (SO2)--OsO4 method was used to examine the distribution of glycoproteins in rabbit fibroblast cells infected with Herpes simplex virus type 1. In non-infected cells, a low level of staining was seen over the plasma membrane and the membranes of the Golgi apparatus. At 17 hr post-infection, the intensity of reaction was increased to include not only a relatively heavy staining of the plasma membrane, including the numerous microvilli characteristic of infected cells, and of the newly proliferated Golgi membranes, but also the envelopes of intracytoplasmic and extracellular virions. A very faint but only occasional staining also was associated with the virus-induced reduplications of the inner nuclear membrane and the envelopes of associated enveloping nucleocapsids. We suggest that such differences in the intensity of staining may be related either to the amount of glycoproteins or to the sequential maturation of the viral glycoproteins. We also observed that the structurally modified portions of the Golgi membranes at the position where intracytoplasmic naked nucleocapsids bud into the Golgi cisternae usually exhibit a more intense reaction for glycoproteins than do the adjacent portions of the Golgi membranes. This supports the evidence for an envelopment of nucleocapsids in the cytoplasm, but it does not indicate whether this event obligatorily follows or only occasionally takes the place of the envelopment of nucleocapsids at the inner nuclear membrane. In either event, the envelopes of all mature virions exhibit a prominent reaction to glycoproteins.

Effects of tunicamycin and monensin on the distribution of highly phosphorylated proteins in cells infected with herpes simplex virus type 1.

New aspects of the distribution of highly phosphorylated proteins in cells infected with herpes simplex virus type 1 (HSV-1) were investigated at the ultrastructural level by the use of drugs which inhibit the glycosylation of viral proteins. The highly phosphorylated proteins were localized by the bismuth tartrate procedure applied on sections of glutaraldehyde-fixed cells embedded in Lowicryl. The drugs employed were tunicamycin, which alters the glycosylation activity of the rough endoplasmic reticulum (RER), and monensin, which blocks the migration of vesicles of the Golgi apparatus (GA) thereby impairing the glycosylation function of the GA. Tunicamycin induced proliferation of RER and the accumulation of highly phosphorylated proteins on its membranes and also impaired GA vesicle maturation and inhibited the usual accumulation of phosphorylated proteins within them. Monensin induced proliferation of the nuclear envelope, including both outer and inner membranes, with bismuth bound to staggered segments of the latter, and also affected the GA in that bismuth-binding proteins were accumulated on the external surface of the swollen vesicles instead of the lumen. These data suggest that an injury of one membrane system, RER or GA, engenders consequential effects on the other. This also supports evidence for an interrelationship between post-translational glycosylation and phosphorylation of proteins in HSV infection.

Visualization of glycoproteins after tunicamycin and monensin treatment of herpes simplex virus infected cells.

The effects of tunicamycin and monensin on the morphogenesis of herpes simplex virus type 1 and on the ultrastructure and function of host cell membranes was investigated by conventional technics of electron microscopy and cytochemical localization of glycoproteins with thiocarbohydrazide-SO2. Infected RS 537 rabbit fibroblasts were treated with tunicamycin, which inhibits the glycosylation of many glycoproteins, or monensin, which inhibits the transport of proteins to the cell surface, and were compared with untreated infected cells. Tunicamycin treatment almost entirely suppresses the perinuclear envelopment of viral capsids, induces the nuclear export of unusually numerous naked viral capsids, and prevents the proliferation of the Golgi apparatus. On the other hand, perinuclear envelopment of viral capsids still occurs following a monensin treatment; however, enveloped viral capsids are not released into the extracellular space; in addition this treatment induces the proliferation of the rough endoplasmic reticulum (RER). The number of structures stained for glycoproteins in tunicamycin-treated cells is markedly lower than that in nontreated infected cells, whereas an unusual additional staining of the entire outer nuclear membrane and of the RER occurs following monensin treatment.

Quantitative variations in nucleolar components within pineal gland cells during the rat estrous cycle.

Quantitative changes in the size of pinealocyte nucleoli have been reported in various studies on this cell type. However, the significance of quantitative changes in the nucleolar components is unknown. The present study is an attempt to analyze ultrastructural and morphometric modifications occurring in the pinealocyte nucleolar components during the estrous cycle in female rats. The fibrillar centers showed an increase during estrus consistent with a decrease in pinealocyte nucleolar activity and melatonin pineal levels. The fibrillar components and granular components tended to display a reciprocal relationship. An increase in the dense fibrillar component took place at metaestrus and diestrus when melatonin synthesis increased in pinealocytes. Maximum values of granular and interstitial components were found at the proestrus phase before the day of ovulation.

Immunocytochemical location of endogenous cytokinins in buds of kiwifruit (Actinidia deliciosa) during the first hours of in vitro culture.

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase as markers for light microscopy or immunoglobulin G-gold for electron microscopy respectively were used for the localization of cytokinins [9-beta-D-ribofuranosil-N6-(delta2-isopentenil) adenina ([9R]iP), 9-beta-D-ribofuranosyl-zeatin ([9R]Z) and 9-beta-D-ribofuranosyl-dihydrozeatin ([9R](diH)Z)] in kiwifruit (Actinidia deliciosa) meristematic cells of the second nodal segment. Immunolocation at the cellular level was carried out in cells from explants grown during 16 and 72 h in liquid medium. Subcellular immunolocalization was performed in cells from explants grown for 35 d on agar solidified-medium and for 30 min, 4 and 16 h in liquid medium with cellulose plugs as explant support. Taken as a whole, the results obtained for Actinidia deliciosa show that the studied cytokinins change their location during the culture period, although they can always be found to a greater or lesser extent in the nucleus and the cytoplasm. For instance, [9R]Z appears in the cytoplasm and in the nucleus during the first hours of culture and later is the only one that appears located mainly in nucleus. On the other hand, [9R](diH)Z changes from being predominantly located in the nucleus to practically appearing only in the cytoplasm at the end of the culture period. [9R]iP is principally found up to 4 h of culture in the cytoplasm, and at 16 h is evenly distributed in all the subcellular compartments except in the chloroplast. The existence of a large amount of cytokinins in the nucleus during the first hours of culture compared with the immunolabelling density at 35 d is probably due to the activation of cell cycle mechanisms leading to organogenic development at the beginning of culture.

Immunodetection of low concentrations of ovalbumin in cryoprocessed quail oviduct cells by semi-automatic quantitative analysis.

In a previous paper (Quintana et al, Biol Cel 72, (1991) 167-180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significant higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.

(3-Cryo) methods (cryofixation, cryosubstitution and cryoembedding) for processing of tissues for ultrastructural and immunocytochemical studies. Application to oviduct cells of laying quail.

Cryomethods occupy a privileged position among the procedures used for the preparation of biological samples for the various studies that may be performed in electron microscopy (ultrastructural, immunocytochemical and microanalysis in situ). In general, cryomethods are specific to one, or a maximum of 2 types of application. The (3-Cryo) methods (cryofixation, cryosubstitution without fixatives and cryoembedding in the new Lowicryl resins (K11M or HM23) are a set of methods for correlating new structural information with analytical and biochemical data. However, these 3-Cryomethods are delicate, complicated and expensive. To demonstrate that they can be performed, at least in part, with home-made systems at a reasonable cost, we have carried out a structural and immunocytochemical study on the oviduct of the laying quail. We studied the localization of 2 proteins, one cytoplasmic (ovalbumin) and the other nucleolar (B-36). The results provided by the 3-Cryomethods are compared with those obtained with other immunocytochemical methods, including tissue processed by conventional chemical fixation and high or low temperature embedding, or by 2-Cryomethods (cryofixation and cryosubstitution).