Binding of lectins to DNA micro-assemblies: modification of nucleo-cages with lactose-conjugated psoralen

Spherical DNA micro-assemblies appended with lactose units (Lactose-nucleo-cages, Lac-NC) are newly developed. DNA spherical assemblies self-assembled from suitably designed three oligodeoxyribonucleotides (ODNs) 1-3 were cross-linked by lactose-conjugated psoralen derivative 4. Confocal laser scanning fluorescence microscopy (CLSM) observation of Lac-NC shows that rhodamine labeled peanut lectin (Rho-PNA: a galactose-specific lectin) binds to lactose-modified nucleo-cages with higher affinity compared to that of unmodified nucleo-cages. Binding isotherm experiments indicated that the apparent affinity constant of Rho-PNA to Lac-NC was in the order of 10(5) M(-1).     

Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development

During the 3-h developmental stage 14 in the chick, intercellular transfer of iontophoresed fluorescent dyes becomes less sensitive to the lowering of intracellular pH by either CO2 or acetate ions. Up to developmental state 14, dye transfer between lens cells is reversibly blocked by exposure to 50% CO2. Beyond stage 14, dye transfer between these cells is no longer reversibly blocked by elevated pCO2. Electronic coupling is present throughout lens development and is not reversibly blocked by high pCO2 at any stage. The gap junctions joining the lens cells show morphological changes at developmental stage 14. Up to stage 14, all gap junctions observed between chick lens cells have connexon assemblies that appear condensed or crystalline following routine freeze-fracture microscopy. Beyond stage 14, chick lens cells express gap junctions with both the condensed assemblies and the dispersed assemblies characteristic of adult lens gap-junction structure.     

Integrin-targeting block copolymer probes for two-photon fluorescence bioimaging

Targeted molecular imaging with two-photon fluorescence microscopy (2PFM) is a powerful technique for chemical biology and, potentially, for noninvasive diagnosis and treatment of a number of diseases. The synthesis, photophysical studies, and bioimaging are reported for a versatile norbornene-based block copolymer multifunctional scaffold containing biocompatible (PEG), two-photon fluorescent dyes (fluorenyl) and targeting (cyclic-RGD peptide) moieties. The two bioconjugates, containing two different fluorenyl dyes and cRGDfK covalently attached to the polymer probe, formed a spherical micelle and self-assembled structure in water, for which size was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cell viability and 2PFM imaging of human epithelial U87MG cell lines that overexpress α(v)β(3) integrin was performed via incubation with the new probes, along with negative control studies using MCF-7 breast cancer cells and blocking experiments. 2PFM microscopy confirmed the high selectivity of the biocompatible probe in the integrin-rich area in the U87MF cells while blocking as well as negative control MCF-7 experiments confirmed the integrin-targeting ability of the new probes.     

Microstructures of an amelogenin gel matrix.

The thermo-reversible transition (clear <--> opaque) of the amelogenin gel matrix, which has been known for some three decades, has now been clarified by microstructural investigations. A mixed amelogenin preparation extracted from porcine developing enamel matrix (containing "25K," 7.4%; "23K," 10.7%; "20K," 49.5%; and smaller peptides, 32.4%) was dissolved in dilute formic acid and reprecipitated by adjusting the pH to 6.8 with NaOH solution. Amelogenin gels were formed in vitro by sedimenting the precipitate in microcentrifuge tubes. The gels were fixed with Karnovsky fixative at 4 and 24 degrees C, which was found to preserve their corresponding clear (4 degrees C) and opaque (24 degrees C) states. Scanning electron microscopy, atomic force microscopy, and transmission electron microscopy were employed for the microstructural characterization of the fixed clear and opaque gels. The amelogenin gel matrix was observed to possess a hierarchical structure of quasi-spherical amelogenin nanospheres and their assemblies. The nanospheres of diameters 8-20 nm assemble to form small spherical assemblies of diameters 40-70 nm that further aggregated to form large spherical assemblies of 70-300 nm in diameter. In the clear gel, most of the large assemblies are smaller than 150 nm, and the nanospheres and assemblies are uniformly dispersed, allowing an even fluid distribution among them. In the opaque gel, however, numerous spherical fluid-filled spaces ranging from 0.3 to 7 microm in diameter were observed with the majority of the large assemblies sized 150-200 nm in diameter. These spaces presumably result from enhanced hydrophobic interactions among nanospheres and/or assemblies as the temperature increased. The high opacity of the opaque (24 degrees C) gel apparently arises from the presence of the numerous fluid-filled spaces observed compared to the low-temperature (4 degrees C) preparation. These observations suggest that the hydrophobic interactions among nanospheres and different orders of amelogenin assemblies are important in determining the structural integrity of the dental enamel matrix.     

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes

OBJECTIVE: To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. STUDY DESIGN: Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. RESULTS: Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. CONCLUSION: Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.     

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.     

The formation of a novel supramolecular structure by amyloid of poly-L-glutamic acid

Polyglutamic acid (PE) has been shown to form amyloid fibrils in vitro under pH value of 4.0. However, under the pH of 2.0, a further self-association process resulting in a novel supramolecular structure was observed. These supramolecular assemblies had diameters ranging from 1 to 20 μm and lengths up to several hundred microns, which were significantly larger than those of typical “amyloid fibrils”. The existence of amyloid-like structure within these assemblies was confirmed with Fourier transform infrared spectroscopy and Thioflavin T fluorescence assay. The aggregation process of PE was studied by direct observation of electronic microscopy. The supramolecular assemblies appeared to be formed in a hierarchical process in which the preformed amyloid-like subunits self-assembled into higher-order assemblies in a well-organized pattern.     

Computational identification of three-way junctions in folded RNAs: a case study in Arabidopsis

Three-way junctions in folded RNAs have been investigated both experimentally and computationally. The interest in their analysis stems from the fact that they have significantly been found to possess a functional role. In recent work, three-way junctions have been categorized into families depending on the relative lengths of the segments linking the three helices. Here, based on ideas originating from computational geometry, an algorithm is proposed for detecting three-way junctions in data sets of genes that are related to a metabolic pathway of interest. In its current implementation, the algorithm relies on a moving window that performs energy minimization folding predictions, and is demonstrated on a set of genes that are involved in purine metabolism in plants. The pattern matching algorithm can be extended to other organisms and other metabolic cycles of interest in which three-way junctions have been or will be discovered to play an important role. In the test case presented here with, the computational prediction of a three-way junction in Arabidopsis that was speculated to have an interesting functional role is verified experimentally.     

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system.Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy.Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates.Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks.     

Designed aromatic homo-dipeptides: formation of ordered nanostructures and potential nanotechnological applications

Molecular self-assembly offers new routes for the fabrication of novel materials at the nano-scale. Peptide-based nanostructures represent nano-objects of particular interest, as they are biocompatible, can be easily synthesized in large amounts, can be decorated with functional elements and can be used in various biological and non-biological applications. We had previously revealed the formation of highly ordered tubular structures by the diphenylalanine peptide, the core recognition motif of Alzheimer's beta-amyloid polypeptide, due to specific aromatic interactions. We further confirmed this model and demonstrated that a non-charged peptide analogue, Ac-Phe-Phe-NH2, self-assembled into similar tubular structures. We later explored other amine and carboxyl modified diphenylalanine peptide analogues and revealed that these dipeptides can form ordered tubular structures at the nanometric scale. Moreover, a very similar peptide, the diphenylglycine, self-assembled into ordered nano-spherical assemblies. Here we extend our research and explore the self-assembly of other homo-aromatic dipeptides in which their phenyl side-chains are modified with halogen atoms (di-para-fluoro-Phe, di-pentafluoro-Phe, di-para-iodo-Phe), additional phenyl groups (di-4-phenyl-Phe), or with nitro substitutions (di-para-nitro-Phe). We also probed the effect of the alteration of the phenyl groups with naphtyl groups (di-D-1-Nal and di-D-2-Nal). In all cases, well-ordered nanostructures were obtained and studied by scanning electron microscopy, transmission electron microscopy and vibrational spectroscopy. Taken together, the current work and previous ones define the homo-aromatic dipeptide as a central motif for the formation of ordered self-assembled tubular, spherical and two-dimensional structures at the nano-scale.     

Calcium-induced assembly of adherens junctions in keratinocytes

Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin-containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes.     

Disturbed structural interactions between microfilaments and tight junctions in rat hepatocytes during extrahepatic cholestasis induced by common bile duct ligation

 Microfilaments in epithelial cells are important for the structural and functional integrity of tight junctions. In the present study, we examined the relationship between microfilaments and tight junctions in hepatocytes of rat liver following common bile duct ligation (CBDL) for up to 2 weeks. Actin filaments and tight junctions were studied by fluorescence microscopy using 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph) and an anti-ZO-1 antibody, respectively. Double-stained sections were examined with confocal laser scanning microscopy (CLSM). Electron microscopy was applied for the assessment of structural alterations in microfilaments and in tight junctions with detergent-extraction and freeze-fracture preparations. Our results showed that F-actin was present at the entire plasma membrane of hepatocytes in control liver, whereas CBDL increased the amount of F-actin mainly at the bile canalicular and lateral plasma membranes. Simultaneously, the immunofluorescence of ZO-1 underwent striking changes, i.e., from a uniform to an irregular staining pattern with various fluorescence intensities. CLSM demonstrated a colocalization of ZO-1 and F-actin in control liver and its deterioration in CBDL liver. Electron microscopy showed marked alterations of microfilaments and tight junctions due to CBDL. It is concluded that actin filaments are intimately associated with tight junctions in normal hepatocytes. CBDL impairs this association by progressively diminishing the structural interaction between F-actin and ZO-1, which may in turn lead to functional disturbances of tight junctions. Accepted: 28 August 1996     

Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells.

The intracellular distribution and metabolism of microinjected fluorescently-labeled oligonucleotides (ODNs) have been evaluated using confocal fluorescence microscopy. Fluorescent phosphodiester ODNs, microinjected into the cytoplasm of mammalian cells, rapidly accumulate within the nucleus; the fluorescence disappears with a half-life of 15-20 minutes. Microinjected fluorescent phosphorothioate ODNs remain in the nucleus for more than 24 hours. The persistence of fluorescence depends on the length of the ODN. Modification of the 3' end of phosphodiester ODNs does not significantly slow the rapid disappearance of fluorescence, although certain 3' modifications localize ODNs into the cytoplasm. Using specially designed ODNs, endonuclease activity is demonstrated to exist in the cytoplasm and nucleus. Modification of the 2' position of the ribose rings of a fluorescent phosphodiester oligodeoxynucleotide with O-methyl or O-allyl does not alter its intracellular distribution; however, the 2'-O-allyl modification stabilizes the persistence of fluorescence more than 60-fold compared to the 2'-deoxy control. Thus, the experiments indicate that somatic cells contain nucleolytic activities which degrade microinjected ODNs; however, chemical modification can dramatically circumvent this process.     

Histochemical and ultrastructural features of the epidermis of the land planarian Bipalium adventitium

The epidermis of the land planarian Bipalium adventitium was examined by light and electron microscopy. In all regions, the epidermis consists of a simple columnar ciliated epithelium associated with a prominent basement membrane. The epithelial cells, possessing abundant microvilli and poorly developed terminal webs, are conjoined laterally at their apical ends by septate junctions. The epidermis of the creeping sole is distinguished from that of adjoining regions by a “insunken” condition of the epithelial cells, a greater number of cilia per cell, and an absence of glandular secretions other than mucus. The insunken cells of the sole possess large glycogen disposits and attributes of metabolically active cells. Unusual intranuclear inclusions of unknown significance are also found in many of the epidermal cells in all regions. The basement membrane lacks distinct layering and consists of fine fibrils displaying a beaded appearance but no obvious cross-banding. Histochemical tests indicate that the fibrils are collagenous. In addition to mucus, secretory material found in nonsole regions includes lamellated granules and rhabdites, both stained intensely by acidic dyes. Rhabdites and the basement membrane also contain disulfide-enriched proteins. In scanning electron micrographs, the sole appears as a faint, longitudinally oriented band extending along the entire length of the animal. In all regions except the sensory border of the head, the microvilli are generally obscured by the densely arranged cilia. The sensory border consists of a row of toothlike papillae and grooves covered almost exclusively by microvilli, small club-shaped structures, and larger spherical protrusions.     

Polymer-stabilized phospholipid vesicles formed on polyelectrolyte multilayer capsules

Phospholipid vesicles on polyelectrolyte multilayer shells can be stabilized against ethanol by coating a single cationic polyelectrolyte. Confocal laser scanning microscopy (CLSM) proved that the lipids were stabilized by cationic polyelectrolytes and the permeability to small hydrophilic dyes was decreased. Measurements of fluorescence recovery after photo-bleaching (FRAP) with individual capsules enable quantification of release profiles.     

Pleated septate junctions in leech photoreceptors: ultrastructure, arrangement of septa, gate and fence functions

The leech photoreceptor forms a unicellular epithelium: every cell surrounds an extracellular “vacuole” that is connected to the remaining extracellular space via narrow clefts containing pleated septate junctions. We analyzed the complete structural layout of all septa within the junctional complex in elastic brightfield stereo electron micrographs of semithin serial sections from photoreceptors infiltrated with colloidal lanthanum. The septa form tortuous interseptal corridors that are spatially continuous, and open ended basally and apically. Individual septa seem to be impermeable to lanthanum; interseptal corridors form the only diffusional pathway for this ion. The junctions form no diffusion barrier for the electron-dense tracer Ba²⁺, but they hinder the diffusion of various hydrophilic fluorescent dyes as demonstrated by confocal laser scanning microscopy (CLSM) of live cells. Even those dyes that penetrate gap junctions do not diffuse beyond the septate junctions. The aqueous diffusion pathway within the septal corridors is, therefore, less permeable than the gap-junctional pore. Our morphological results combined with published electrophysiological data suggest that the septa themselves are not completely tight for small physiologically relevant ions. We also examined, by CLSM, whether the septate junctions create a permeability barrier for the lateral diffusion of fluorescent lipophilic dyes incorporated into the peripheral membrane domain. AFC₁₆, claimed to remain in the outer membrane leaflet, does not diffuse beyond the junctional region, whereas DiIC₁₆, claimed to flip-flop, does. Thus, pleated septate junctions, like vertebrate tight junctions, contribute to the maintenance of cell polarity.     

Preparation and physicochemical characteristics of self-assembled nanoparticles of deoxycholic acid modified-carboxymethyl curdlan conjugates

Various deoxycholic acid (DOCA)-modified-carboxymethylated (CM)-curdlan (DCMC) were synthesized and characterized by FTIR, ¹H NMR and XRD. The degree of DOCA substitution (DS), as spectrophotometrically determined, was 2.1, 3.2, 4.1, or 6.3 DOCA groups per hundred sugar residues of CM-curdlan. The physicochemical properties of the self-assembled nanoparticals in aqueous media were investigated using ¹H NMR, dynamic light scattering, zeta potential, transmission electron microscopy (TEM) and fluorescence spectroscopy. DCMC conjugates provided monodispersed self-assembled nanoparticles in water, with mean diameter decreasing from 192 to 347 nm with DOCA DS increasing. Moreover, the mean diameter also increased with decreasing pH in PBS. Zeta potential of DCMC self-assembled nanoparticles exhibited near −60 mV in distilled water and −26 to −36 mV in PBS, indicating these nanoparticles were covered with negatively charged CM-curdlan shells. The critical aggregation concentration (cac) of the DCMC were dependent on the degree of substitution (DS) of DOCA and were slightly lower in PBS than in distilled water. The TEM images demonstrated that these self-assembled nanoparticles were of spherical shape.     

Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells

The macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid-phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC-dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid-phase endocytosis and that microfilaments hinder this process. © 1993 Wiley-Liss, Inc.     

Use of confocal laser scanning microscopy in systematics of insects with a comparison of fluorescence from different stains

Abstract Confocal laser scanning microscopy has become a valuable tool for a wide range of investigations in the biological sciences, but its use in insect systematics has been neglected. Confocal microscopy depends on the degree of fluorescence of the examined specimens, which is aided either by fluorescent dyes or autofluorescence of the specimen. This study provides methods for using a combination of fluorescent dyes and autofluorescence to provide images that document the value of confocal microscopy for systematic research with insects. Fluorescence was compared from Lepidoptera genitalia dissections that were unstained or stained with merbromin (mercurochrome), safranine, chlorazol black E, eosin Y, eosin Y + chlorazol black E, and orange‐G. The unstained specimen showed that chitin autofluorescences to a small degree. The comparison of stains showed that use of eosin Y provides the best images, followed by safranine and mercurochrome. Orange‐G and chlorazol black are the least fluorescent and provide poor images, even when chlorazol black is combined with eosin.