Wall Vesicles Research Articles

Hypoglycemic effects of mountain caviar extract and inhibitory mechanism of saponins, including momordin Ic, on glucose absorption.

Mountain caviar is a fruit of Kochia scoparia that contains momordin Ic as a major saponin constituent. Its extract (MCE) has been shown to suppress blood glucose elevations in the human oral glucose tolerance test (OGTT) as well as increases in blood glucose in OGTT, gastric emptying (GE), and glucose incorporation in the small intestine in rats. However, the effects of MCE and momordin Ic on glucose absorption in mice and these action mechanisms have not been examined for more than 2 decades. Therefore, we herein investigated the effects of MCE, its saponin fraction, and momordin Ic on blood glucose elevations in mice. Mouse blood glucose elevation tests were performed on carbohydrate-loaded mice. The mountain caviar saponin fraction significantly delayed blood glucose elevations in glucose-, sucrose-, and soluble starch-loaded mice. In glucose-loaded mice, the saponin fraction, MCE, and momordin Ic significantly suppressed rapid glucose elevations after glucose loading, but not sucrose loading. A mouse GE study was performed by loading with glucose and phenolphthalein solution. Momordin Ic and MCE strongly suppressed mouse GE. Intestinal glucose absorption was evaluated by the incorporation of 2-deoxyglucose (2-DG) into Caco-2 cell layers and mouse duodenum wall vesicles. The results obtained showed that momordin Ic inhibited the incorporation of 2-DG into Caco-2 cells and mouse duodenum vesicles. Collectively, these results suggest that MCE, particularly the principal saponin, momordin Ic, preferably suppressed glucose-induced blood glucose elevations and delayed carbohydrate-induced glucose elevations in mice. The underlying mechanism was found to involve the suppression of GE and intestinal glucose absorption.

Synthesis of multilamellar walls vesicles polyelectrolyte-surfactant complexes from pH-stimulated phase transition using microbial biosurfactants

Multilamellar wall vesicles (MLWV) are an interesting class of polyelectrolyte-surfactant complexes (PESCs) for wide applications ranging from house-care to biomedical products. If MLWV are generally obtained by a polyelectrolyte-driven vesicle agglutination under pseudo-equilibrium conditions, the resulting phase is often a mixture of more than one structure. In this work, we show that MLWV can be massively and reproductively prepared from a recently developed method involving a pH-stimulated phase transition from a complex coacervate phase (Co). We employ a biobased pH-sensitive microbial glucolipid biosurfactant in the presence of a natural, or synthetic, polyamine (chitosan, poly-l-Lysine, polyethylene imine, polyallylamine). In situ small angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) show a systematic isostructural and isodimensional transition from the Co to the MLWV phase, while optical microscopy under polarized light experiments and cryo-TEM reveal a massive, virtually quantitative, presence of MLWV. Finally, the multilamellar wall structure is not perturbed by filtration and sonication, two typical methods employed to control size distribution in vesicles. In summary, this work highlights a new, robust, non-equilibrium phase-change method to develop biobased multilamellar wall vesicles, promising soft colloids with applications in the field of personal care, cosmetics and pharmaceutics among many others.

Mycobacterium avium Complex Extracellular Vesicles Attenuate Inflammation via Inducing IL-10.

Mycobacterium avium complex (MAC) is an ubiquitous acid-fast bacterium. MAC cell wall and membrane release extracellular vesicles (EVs) into different media. The immunogenic effects of EVs isolated from MAC remain unknown. The aim of this study was to determine the EVs effect on macrophage cytokine production. MAC EVs were extracted and purified using differential centrifuges also known as Claassen’s method, with some modifications. After protein analysis of EVs, and scanning electron microscopy (SEM), the EVs were injected into BALB/c mice for in vivo experiments. The concentration of interferon gamma (IFN-γ) and interleukin 10 (IL-10) in the spleen immune cell culture was measured by sandwich ELISA. We for the first time showed that MAC can naturally produce EVs. The extraction method was technically-feasible, efficient and affordable. The SEM analysis showed that EVs diameter was similar to other studies on mycobacteria, and EVs maintained their spatial characterization. The results of the cytokine assays indicated that EV-treated cells secreted IL-10 (P = 0.034) but not IFN-γ (P = 0.037). Our findings suggest that EVs of M. avium could have anti-inflammatory effects. They can be used as a suppressor or regulator of inflammation via IL-10. The replication of the anti-inflammatory response of MAC EVs in future studies may indicate a new therapeutic agent for inflammation.

Shear Induced Morphological Transformation of Large Compound Micelles Formed by Glutathione End‐capped Poly(4‐vinylpyridine)

AbstractHere we report novel shear‐sensitive polymeric large compound micelles (LCMs) formed by self‐assembly of glutathione end‐capped poly(4‐vinylpyridine) (GP4VP) in water containing 4 vol% methanol. The amphiphile prefers to form the kinetically stabilized LCMs due to the small length ratio of the hydrophilic part to the hydrophobic part; the encapsulation of the hydrophilic part within LCMs causes only a small enthalpy loss from a thermodynamic view. Sheared by vortex flows generated by vigorous stir, the LCMs evolve to thick wall vesicles, thin wall vesicles, broken vesicles and "pearl‐necklace" structures consecutively. The high flexibility of the LCMs and the internal tension caused by the encapsulation of the hydrophilic part are responsible for the high shear sensitivity of the LCMs.

Well-Controlled Formation of Nanofibers and Double Wall Vesicles through the Electrostatic-Assisted Assembly of a Couple of Star Polyelectrolytes-Complementary

A series of water-soluble hexa-armed cationic star polyelectrolytes and anionic star polyelectrolytes with a hexafunctional cyclotriphosphazene derivative core were synthesized. The self-assembly behaviors of these complementary star polyelectrolyte pairs in water in the effect of electrostatic interaction were investigated. It was found that the complementary star polyelectrolyte pairs system could form various morphologies such as fiber bundles and double wall vesicles in water by adjusting molecular weight, polymer concentration, the matching degree of star polyelectrolyte pairs, and the preparation conditions of the self-assembly solvents. The self-assembly mechanisms for the formation of nanofibers and double wall vesicles were discussed.

Pollen Tube Growth Oscillations and Intracellular Calcium Levels Are Reversibly Modulated by Actin Polymerization

Prevention of actin polymerization with low concentrations of latrunculin B (Lat-B; 2 nm) exerts a profound inhibitory effect on pollen tube growth. Using flow-through chambers, we show that growth retardation starts after 10 min treatment with 2 nm Lat-B, and by 15 to 20 min reaches a basal rate of 0.1 to 0.2 microm/s, during which the pollen tube exhibits relatively few oscillations. If treated for 30 min, complete stoppage of growth can occur. Studies on the intracellular Ca(2+) concentration indicate that the tip-focused gradient declines in parallel with the inhibition of growth. Tubes exhibiting nonoscillating growth display a similarly reduced and nonoscillating Ca(2+) gradient. Studies on the pH gradient indicate that Lat-B eliminates the acidic domain at the extreme apex, and causes the alkaline band to move more closely to the tip. Removing Lat-B and returning the cells to control medium reverses these effects. Phalloidin staining of F-actin reveals that 2 nm Lat-B degrades the cortical fringe; it also disorganizes the microfilaments in the shank causing the longitudinally oriented elements to be disposed in swirls. Cytoplasmic streaming continues under these conditions, however the clear zone is obliterated with all organelles moving into and through the extreme apex of the tube. We suggest that actin polymerization promotes pollen tube growth through extension of the cortical actin fringe, which serves as a track to target cell wall vesicles to preferred exocytotic sites on the plasma membrane.

Are pretreatment simulation estimates predictive of actual rectal and bladder doses delivered during radiation for prostate cancer

Materials/Methods: 15 patients (pts) with localized prostate (pros) cancer were enrolled on a pilot study. Each pt was scanned 25 times during the course of RT (once at simulation with a full bladder and empty rectum and 24/42 tmt days) for a total of 369 evaluated CTs. On each scan the bladder (bld), seminal vesicles, pros, rectum and rectal wall were contoured. An IMRT plan was generated for each pt on the initial simulation (sim) CT. This plan was then superimposed on each of the 24 treatment (tmt) CT scans. Isocentric fiducials were used to align tmt plans after which the clinically utilized BAT shift was applied. DVHs were generated for rectal anatomic volume (rav), rectal wall (rw), bld, prostate and seminal vesicles (prsv). For the purposes of this evaluation we looked in detail at 3 pts (75 CTs). The choice of pts was as follows: the pt with the largest variation in pros center of volume (COV) over the RT course, the pt with the least variation, and one with average daily pros COV change.

Antimicrobial activity of magainin analogues against anaerobic oral pathogens

Magainins are a family of potent antimicrobial cationic peptides that possess antimicrobial activity against a wide range of target organisms. In this study, the antimicrobial activity of synthetic magainin–mimetic compounds MSI-751 and MSI-774 was investigated against the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Eikenella corrodens, Prevotella loescheii and Prevotella intermedia. P. gingivalis was more susceptible to MSI-751 than to MSI-774, whereas the other oral pathogens showed little difference in susceptibility to the two compounds. MSI-751 exhibited a rapid, dose-dependent bactericidal effect on P. gingivalis. Electron microscopy of MSI-751-treated P. gingivalis revealed intact cell wall vesicles devoid of cell contents, suggesting perturbation of the cytoplasmic membrane by this compound, perhaps equivalent to formation of membrane-disruptive ion channels by magainin peptides. These studies demonstrate that synthetic magainin derivatives exhibit antimicrobial activity against oral pathogens by disruption of cell membrane integrity.

Ca2+ Shuttling in Vesicles During Tip Growth in Neurospora crassa

Tip-growing organisms maintain an apparently essential tip-high gradient of cytoplasmic Ca2+. In the oomycete Saprolegnia ferax, in pollen tubes and root hairs, the gradient is produced by a tip-localized Ca2+ influx from the external medium. Such a gradient is normally dispensable for Neurospora crassa hyphae, which may maintain their Ca2+ gradient by some form of internal recycling. We localized Ca2+ in N. crassa hyphae at the ultrastructural level using two techniques (a) electron spectroscopic imaging of freeze-dried hyphae and (b) pyroantimoniate precipitation. The results of both methods support the presence of Ca2+ in the wall vesicles and Golgi body equivalents, providing a plausible mechanism for the generation and maintenance of the gradient by Ca2+ shuttling in vesicles to the apex, without exogenous Ca2+ influx. Ca2+ sequestration into the vesicles seems to be dependent on Ca2+–ATPases since cyclopiazonic acid, a specific inhibitor of Ca2+ pumps, eliminated all Ca2+ deposits from the vesicles of N. crassa.

A Tip-High Gradient of a Putative Plasma Membrane SNARE Approximates the Exocytotic Gradient in Hyphal Apices of the Fungus Neurospora crassa

Gupta, G. D., and Heath, I. B. 2000. A tip-high gradient of a putative plasma membrane SNARE approximates the exocytotic gradient in hyphal apices of the fungus Neurospora crassa. Antibodies to the Saccharomyces cereviseae plasma membrane t-SNARE Sso2p identify a putative 39-kDa homologue in Neurospora crassa. The 39-kDa protein is enriched in plasma membrane (PM) and occurred with other membranes. It is extractable by detergent, but not chaotropic or alkali agents, suggesting membrane insertion. Immunoprecipitation with anti-Sso2p coprecipitated a ∼100-kDa, Mg+-ATP-sensitive band with the 39-kDa protein, suggesting a ternary SNARE complex. Affinity-purified anti-Sso2p gave hyphal staining patterns most consistent with protein localization on both the PM and intracellular exocytotic apical wall vesicles. The PM staining in hyphal apices formed a tip-high gradient, not as steep as that predicted by the “hyphoid equation,” but closer to published gradients of cell wall matrix deposition. We conclude that the t-SNAREs are transported to the PM on the apical vesicles, but their tip-high gradient alone is insufficient to explain the vesicle fusion gradient in growing tips.

Aggregation and collapse of fungal wall vesicles in hyphal tips: a model for the origin of the Spitzenkörper

The intracellular origins of polarity and branch initiation in fungi centre upon a localization in the supply of fungal wall constituents to specific regions on the hyphal wall. Polarity is achieved and maintained by accumulating secretory vesicles, prior to incorporation into the wall, in the form of an apical body or Spitzenkörper. However, neither the mechanisms leading to this accumulation nor the initiation of branching, are as yet understood. We propose a mechanism, based on experimental evidence, which considers the mechanical properties of the cytoskeleton in order to explain these phenomena. Cytoskeletal viscoelastic forces are hypothesized to be responsible for biasing vesicles in their motion, and a mathematical model is derived to take these considerations into account. We find that, as a natural consequence of the assumed interactions between vesicles and cytoskeleton, wall vesicles aggregate in a localized region close to the tip apex. These results are used to interpret the origin of the Spitzenkörper. The model also shows that an aggregation peak can collapse and give rise to two new centres of aggregation coexisting near the tip. We interpret this as a mechanism for apical branching, in agreement with published observations. We also investigate the consequences and presumptive role of vesicle—cytoskeleton interactions in the migration of satellite Spitzenkörper. The results of this work strongly suggest that the formation of the Spitzenkörper and the series of dynamical events leading to hyphal branching arise as a consequence of the bias in vesicle motion resulting from interactions with the cytoskeleton.

Herpes simplex virus type 2 infection in a 5-year-old boy presenting with recurrent chest wall vesicles and a possible history of herpes encephalitis

A 5-year-old hyperkinetic but otherwise healthy child presented with recurrent irritable vesicles and erosions of the anterior chest wall; they have been apparent since the age of 15 months. Wound swab cultures yielded herpes simplex virus type-2 (HSV-2) and Western blot serology showed past exposure to both HSV-1 and HSV-2. Skin biopsy results further supported a herpes virus infection. Magnetic resonance imaging of the brain showed right temporal lobe atrophy. An evaluation showed no evidence of sexual abuse in the patient but a Western blot assay of the mother's serum for HSV-2 was positive, while the father's was negative. In view of the diagnosis of HSV-2 infection in such a young patient, the possible routes of transmission and the time of acquisition of infection were explored. We believe the most likely route of infection in this child was postnatal, through intimate contact with the mother.

Herpes simplex virus type 2 infection in a 5-year-old boy presenting with recurrent chest wall vesicles and a possible history of herpes encephalitis

Herpes simplex virus type 2 infection in a 5-year-old boy presenting with recurrent chest wall vesicles and a possible history of herpes encephalitis

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

A polyclonal antibody against β-1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.

The dynamic behavior of cytoplasmic F-actin in growing hyphae

A dynamic population of cytoplasmic F-actin was observed with electroporated rhodamine phalloidin (RP) staining in growing hyphae ofSaprolegnia ferax. This central actin population was distinct from the fibrillar peripheral network previously described in chemically fixed hyphae in that it was diffuse, pervaded the entire cytoplasm and was most concentrated in the central cytoplasm 8.4 μm from the tip. The peripheral network did not stain with electroporated RP. The apical concentration of central cytoplasmic actin was only present in growing hyphae and developed prior to tip extension. It co-localized with the polarized distribution of mitochondria and endoplasmic reticulum in the tip, suggesting that it functions in positioning these organelles during tip growth. Within the central actin there was a consistent apical cleft which only occurred in growing hyphae and whose position predicted the direction of tip growth. This cleft was coincident with the known accumulation of apical wall vesicles, suggesting that it is either established by vesicle exclusion of the central actin network or is permeated by a portion of the in vivo unstained peripheral network. Photobleaching studies showed that in both growing and non-growing hyphae, cytoplasmic actin continually and rapidly moved from subapical regions to the tip where it accumulated. It mostly moved forward at the rate of tip growth, while some also left the tip, presumably to populate subapical regions.

Vesicle production and fusion during lobe formation inMicrasterias visualized by high-pressure freeze fixation

Two different types of Golgi vesicles involved in wall formation can be visualized during lobe growth inMicrasterias when using high-pressure freeze fixation followed by freeze substitution. One type that corresponds to the “dark vesicles” (DV) described in literature seems to arise by a developmental process occurring at the Golgi bodies with the single vesicles being forwarded from one cisterna to the next. The other vesicle type appears to be produced at thetrans Golgi network without any visible precursors at the Golgi cisternae. A third type of vesicle, produced by median andtrans cisternae, contains slime; these are considerably larger than those previously mentioned and they do not participate in wall formation. The distribution of the two types of cell wall vesicles at the cell periphery and their fusion with the plasma membrane are shown for the first time, since chemical fixation is too slow to preserve a sufficient number of vesicles in the cortical cytoplasm. The results indicate that fusions of both types of vesicles with the plasma membrane are possible all over the entire surface of the growing half cell. However, the DVs are much more concentrated at the growing lobes, where they form queues several vesicles deep behind zones on the plasma membrane thought to be specific fusion sites. The structural observations reveal that the regions of enhanced vesicle fusion correspond in general to the sites of calcium accumulation determined in earlier studies. By virtue of the absence of the DVs in the region of cell wall indentations the second type of wall forming vesicle appears prominent; they too fuse with the plasma membrane and discharge their contents to the wall.

Cytoplasmic reorganization accompanies the deposition of a bipolar cell wall in the large-celled red alga Anotrichium tenue

The filamentous red alga Anotrichium tenue C. Aghard (Naegeli) (formerly Griffithsia tenuis C. Aghard; Baldock, 1976, Aust. T. Bot. 24, 509-593) has large (1-2 mm long), cylindrical, multinucleate cells that exhibit a daily, cyclic redistribution of chloroplasts. Chloroplasts accumulate in the mid-region of each growing cell during the day; consequently, filaments appear banded with a light apical end-band, a dark mid-band and a light basal end-band in each growing cell. Chloroplasts disperse at night so that the bands are no longer visible and the cells appear evenly pigmented. Anotrichium tenue also has a type of cell elongation, known as bipolar band growth, in which new material is added to the microfibrillar part of the wall in bands located at the apical and basal poles of elongating cells. This site of wall growth corresponds to the position of the light-colored end-bands present during the day. Here we examine the structural relationship between the cytoplasmic bands and the wall-growth bands. Our results show that, in addition to the previously described bipolar wall bands, there is a non-microfibrillar wall band in the mid-region of the cell. This wall component apparently branches from near the top of the microfibrillar outer wall and terminates near but not at the bottom of the cell. It contains nodules of sulphated polysaccharide material secreted from a band of vesicles, which co-localize with the chloroplasts in the mid-band. The outer wall appears to enclose the entire cell. Nuclei do not redistribute with the chloroplasts or wall vesicles into the mid-band but remain evenly distributed throughout the cytoplasm. Each wall component grows by a different mechanism. We show that two types of wall growth, diffuse and the bipolar-type of tip growth, occur in the same cell and we propose that the observed segregation of the cytoplasm supports localized growth of the unique inner wall component. Additionally, we show that A. tenue is an excellent model for study of the role and mechanism of cytoplasmic compartmentalization and cell polarity during plant cell growth.

A comparison of fluorescent membrane probes in hyphal tips of Saprolegnia ferax

Hyphae of the oomycete Saprolegnia ferax were stained with diverse neutral, anionic, cationic, and phospholipid analog fluorescent membrane probes. Apparently only probes smaller than 1000 Da enter the living hyphae but these permitted near normal growth rates, thus demonstrating their use as vital fungal stains. Collectively the penetrant probes stained all identifiable membranes, but individually none faithfully revealed the total cellular membranes. However, several probes produced a diffuse sub-apical concentration of stain which could not be correlated with any specific membrane type but which may indicate the highest concentration of total cellular membranes. The neutral probes were unique in staining unidentified spherical refractile vesicles. Nile Red was the only probe to show significant staining in the extreme apex where wall vesicles are abundant. DiOC 6 (3) at low concentration was an excellent mitochondria stain as previously reported but at the higher concentrations used in other cells as an endoplasmic reticulum selective stain it was toxic and caused mitochondrial deformation. NDB hexanoic ceramide failed to specifically stain Golgi bodies as previously described in mammalian cells. Our results show that considerable care must be exercised in extrapolating the use of these probes from one cell type to another and in interpreting their staining patterns in new cells.

The organization of tip-growth-related organelles and microtubules revealed by quantitative analysis of freeze-substituted oomycete hyphae

ABSTRACT The distribution of organelles and microtubules in hyphal tips of the oomycete, Saprolegnia feras, were quantitatively determined at high resolution from serial-section electron microscopy of freeze-substituted cells. All the organelles and the microtubules were non-uniformly distributed, each showing a characteristic longitudinal gradient starting at a different point behind the tip. In addition, when the cytoplasmic cross-sectional area was divided into radial regions, all organelles occurred preferentially in either the central (mitochondria and Golgi bodies) or the peripheral (microtubules, wall vesicles and spherical vesicles) region. The nuclei were so large as to span both regions but were always oriented with their centrioles facing the plasmalemma. Microtubules occurred in the extreme tips, became more abundant sub-apically, were predominantly short but increased in mean length with distance from the tip. The correlated patterns of organelle and cytoskeleton organization from this and previous work show that neither the microtubules nor the detected arrays of actin are sufficient to account for most organelle arrangements. However, on the basis of the distribution and orientation of the predominantly elongated wall vesicles, we suggest that the wall vesicles travel radially from their origin at the centrally located Golgi bodies to the cell periphery where they are transported longitudinally to the hyphal tip in conjunction with the plasmalemma-associated actin cables. Our data also suggest that the hyphae contain a cortical ectoplasm with which the nuclei interact, at least in part, via their centrioles and centriole-associated microtubules, and whose mechanical integrity is increased by both the peripheral actin cables and a high density of microtubules. We suggest that the endoplasm is less strong and has physiological properties that enhance the differentiation of endoplasmic reticulum and nuclear envelope into Golgi body production.

Electron spectroscopic imaging (ESI) applied to the detection of potential Ca2+-binding sites in plant cells

The applicability of the electron spectroscopic imaging technique for detection of the intracellular distribution of calcium in plant cells was tested with calyptra cells ofZea mays and with pollen tubes ofLilium longiflorum. After fixation in enhanced Ca(2+) levels and embedding in resin, ultrathin sections were analyzed for the elemental distribution. Calcium and phosphorus were enriched in cell wall, plasma membrane, endoplasmic reticulum, mitochondria, and Golgi vesicles, mainly in granular or globular deposits appearing electron dense in transmission electron microscopy. The results demonstrated that the ESI-technique allows exact localization of calcium enrichment relative to specific cell organelles.