Vesicle Volume Research Articles

Overexpression of synapsin Ia in the rat calyx of Held accelerates short-term plasticity and decreases synaptic vesicle volume and active zone area

Synapsins are synaptic vesicle (SV) proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the SV pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa—known to sustain repetitive transmission in glutamatergic terminals—was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ) alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs.

Dynamics of the Gel to Fluid Phase Transformation in Unilamellar DPPC Vesicles

The dynamics of the gel to fluid phase transformation in 100 nm large unilamellar vesicles (LUV) of 1,2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine (d62-DPPC), has been studied by laser-induced temperature-jump initiation coupled with time-resolved infrared spectroscopy and by MD simulations. The infrared transients that characterize the temperature dependent phase transformation are complex, extending from the nanosecond to the millisecond time scales. An initial fast (submicrosecond) component can be modeled by partial melting of the gel domains, initiated at pre-existing defects at the edges of the faceted structure of the gel phase. Molecular dynamics simulations support the model of fast melting from edge defects. The extent of melting during the fast phase is limited by the area expansion on melting, which leads to a surface pressure that raises the effective melting temperature. Subsequent melting is observed to follow highly stretched exponential kinetics, consistent with collective relaxation of the surface pressure through a hierarchy of surface undulations with different relaxation times. The slowest step is water diffusion through the bilayer to allow the vesicle volume to grow along with its expanded surface area. The results demonstrate that the dominant relaxation in the gel to fluid phase transformation in response to a large T-jump perturbation (compared to the transition width) is fast (submicrosecond), which has important practical and fundamental consequences.

Ultrastructure of Rabbit Embryos Exposed to Hyperthermia and Anti-Hsp 70

The aim of the study was to determine the effect of short-term hyperthermia and Hsp70 blockage on ultrastructural changes in cell organelles and nucleoli of rabbit preimplantation embryos. The embryos were cultured either at 37.5°C (control, C) or 41.5°C (hyperthermia, HT) during 6 h. The antibody against Hsp70 was added into the culture medium (4 μg/ml) of morula stage embryos from C and HT groups. After termination of the culture, the embryos were processed for transmission electron microscopy. The embryos exposed to hyperthermia showed increased volume of lipid droplets, considerable occurrence of cellular debris in the perivitelline space and slight changes in the occurrence of microvilli on the surface of trophoblastic cells. In the embryos exposed to anti-Hsp 70 at 37.5°C, there were considerable changes in mitochondria morphology, decreased volume of dense bodies in the cytoplasm and considerable changes in the occurrence of microvilli on the surface of trophoblastic cells. In the group of embryos exposed simultaneously to hyperthermia and anti-Hsp 70, mitochondria were also expanded and swollen; the volume of flocculent vesicles and lipid droplets was increased and the volume of dense bodies in the cytoplasm was diminished. General organization of the cytoplasm in groups with anti-Hsp70 was characterized by cell organelle segregation. Averaged size of the nucleolar area was significantly increased in the embryos exposed to hyperthermia, whereas in the group exposed to the anti-Hsp70 without hyperthermia it was significantly diminished. Hyperthermia also caused disintegration of compact status of the nucleoli. In presence of anti-Hsp 70, the structural changes, described within the nucleoli during hyperthermia, were not observed. In conclusion, these results document ultrastructural changes in cell organelles of rabbit preimplantation embryo caused by hyperthermia, and also changes in the nucleolar structures, at which presence of Hsp-70 inhibit these changes.

Structure of a micropipette-aspirated vesicle determined from the bending-energy model

The structure of the system consisting of an aspirating pipette and an aspirated vesicle is investigated with fixed total vesicle volume, total vesicle surface area, and aspirated volume fraction, based on the bending-energy model. Through an energetic consideration, the usage of an aspirated volume fraction can be converted to the aspirating pressure for the determination of a phase diagram; the procedure identifies a first-order transition, between a weakly aspirated state and the strongly aspirated state, as the pressure increases. The physical properties of the system are obtained from minimization of the bending energy by an implementation of the simulated annealing Monte Carlo procedure, which searches for a minimum in a multivariable space. An analysis of the hysteresis effects indicates that the experimentally observed aspirating and releasing critical pressures are related to the location of the spinodal points.

Drosophila TRPML Is Required for TORC1 Activation

Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.

Quantification of vesicle characteristics in some diatreme-filling deposits, and the explosivity levels of magma–water interactions within diatremes

Vesicles within juvenile fragments in mafic pyroclastic deposits contain important information about the state of the magma at the time of fragmentation. There have been few vesicle studies of juvenile pyroclasts from mafic phreatomagmatic deposits, however, and none we can find from maar–diatreme volcanoes. In this paper we document the vesicularity and vesicle-population characteristics of juvenile fragments sampled from non-bedded lithified deposits of the Coombs Hills diatreme complex, part of the Ferrar large igneous province, Antarctica. The diatreme-filling pyroclastic deposits, dominated by lapilli tuffs and tuff breccias, contain typically abundant lithic clasts derived mostly from the enclosing sedimentary sequence, and several types of juvenile clasts ranging from blocky to fluidal or “raggy”.In the samples measured, 77–80% of the juvenile pyroclasts ranging in size from 0.5mm to fine lapilli is in the ‘non-vesicular’ to ‘incipiently vesicular’ range (<20% vesicles). Such low vesicularities are expected for pyroclasts from maar–diatreme volcanoes where fragmentation takes place at depth in the diatreme or root zone due to magma–water interaction. A few juvenile clasts, however, are more vesicular, and seven of these were chosen and sectioned for 2D analysis of vesicle shapes and orientation, vesicle number densities (Nv), and vesicle volume distributions. The shapes of the vesicles in the studied sections are mostly elliptical (sometimes polylobate), with mean aspect ratios ranging between 0.67 and 0.72. Circular statistics are used to test for trends in the vesicle long-axis orientation data; non-uniformity of orientations is found in most cases, but the trends are weak. Vesicle volume distributions are often bimodal due to variable coalescence. Total Nv values range from 1.0×102 to 5.7×103mm−3; taking the effects of bubble coalescence into account, these values are similar to those found in pyroclasts from other phreatomagmatic volcanoes, although they also overlap partly with those seen in fire fountain deposits and some basaltic Plinian eruptions.Fluidal- or rag-shaped juvenile clasts, some circular vesicles, and the lack of microlites all suggest that the Coombs Hills magma had a relatively low viscosity prior to fragmentation, despite the basaltic andesite composition. This low viscosity allowed parts of the magma to be fragmented in a non-brittle fashion during phreatomagmatic explosions and to form fluidal clasts. Phreatomagmatic explosions in diatremes can therefore produce diverse types of juvenile clasts simultaneously, and the proportions of each will depend on the explosivity of the magma–water (slurry) interaction and other factors. Recycling of fragments is also thought to be an important factor in generating mixtures of different types of juvenile fragments in diatremes.

Pulling or compressing a vesicle by force: Solution to the bending energy model

The shape transformation induced by pulling or compressing a vesicle along the symmetric axis is discussed in light of the numerical solution to the bending energy model with fixed enclosing volume and surface area. A complete phase diagram in terms of the external force and reduced vesicle volume is investigated. Some experimental observations are explained by the solution.

Extrusion of small vesicles through nanochannels: A model for experiments and molecular dynamics simulations

We propose a model that predicts the final sizes of lipid bilayer vesicles produced by pressure extrusion through nanochannels and we conduct large-scale coarse-grained molecular dynamics simulations of the phenomenon. We show that, to a first approximation independent of pressure, vesicle size can be predicted by a simple geometrical argument that considers an invariable inner vesicle volume enclosed by a finitely extensible lipid bilayer. The pressure dependence is then incorporated in our model by arguing that the effective channel radius decreases with increasing pressure due to a thickening of the lubrication layer between the vesicles and the channel wall. We fit our model to the experimental data of Patty and Frisken [Biophys. J. 85, 996 (2003)]. We predict that at high pressure, vesicle size significantly depends on channel length and, therefore, flow rate. The CGMD simulations reproduce the physical principles of the model. They also show the build-up of the stress in the vesicle, and typical rupture scenarios as the pressure gradient is increased.

Statistical analysis of discrete encapsulation of nanomaterials in colloidal capsules

Encapsulation of materials into capsules becomes discrete at small scales. We propose a measurement and analysis method of discrete encapsulation of nanomaterials in colloidal capsules, with the volume of fL to pL, using the fluorescence flow cytometry. Encapsulation of nano-sized polymer beads and lambda-phage DNA into lipid vesicles (liposomes) is evaluated as a model system. By using fluorescence markers, vesicle volume, membrane quantity and nanomaterials in a large number of individual vesicles are measured simultaneously. We show that the number of encapsulated material (k = 0, 1, 2, …) can be evaluated from the quantized fluorescence intensity distribution, which is then compared with the Poisson statistics. As a result, probability of encapsulating beads and λDNA exhibits characteristic trends that depend on the vesicle volume, lamellarity, and electrostatic interaction, which should reflect the physical interaction in the vesicle formation processes. This measurement and analysis will be a powerful tool in designing the colloidal capsules used in many scientific and industrial applications.

Influence of volatile degassing on initial flow structure and entrainment during undersea volcanic fire fountaining eruptions

Release of dissolved volatiles during submarine fire fountaining eruptions can profoundly influence the buoyancy flux at the vent. Theoretical considerations indicate that in some cases buoyant magma can be erupted prior to fragmentation (~75% vesicle volume threshold). Laboratory simulations using immiscible fluids of contrasting density indicate that the structure of the source flow at the vent depends critically on the relative magnitudes of buoyancy and momentum fluxes as reflected in the Richardson number (Ri). Analogue laboratory experiments of buoyant discharges demonstrate a variety of complex flow structures with the potential for greatly enhanced entrainment of surrounding seawater. Such conditions are likely to favor a positive feedback between phreatomagmatic explosions and volatile degassing that will contribute to explosive volcanism. The value of the Richardson number for any set of eruption parameters (magma discharge rate and volatile content) will depend on water depth as a result of the extent to which the exsolved volatile components can expand.

MRI to assess chemoprevention in transgenic adenocarcinoma of mouse prostate (TRAMP)

BackgroundThe current method to determine the efficacy of chemoprevention in TRAMP mouse model of carcinoma of prostate (CaP) is by extracting and weighing the prostate at different time points or by immunohistochemistry analysis. Non-invasive determination of volumes of prostate glands and seminal vesicles before, during and after treatment would be valuable in investigating the efficacy of newer chemopreventive agents in CaP. The purpose of this study was to determine whether in vivo magnetic resonance imaging (MRI) using a 3 tesla clinical MRI system can be used to follow the effect of chemoprevention in TRAMP model of mouse CaP.MethodsMice were randomized into control and treated groups. The animals in treated group received 10 µmol/kg of CDDO, 5 days a week for 20 weeks. Animals underwent in vivo MRI of prostate gland and seminal vesicles by a clinical 3 Tesla MRI system just before (at 5 weeks), during and at the end of treatment, at 25 weeks. T1-weighted and fat saturation (FATSAT) multiecho fast spin echo T2- weighted images (T2WI) were acquired. Volume of the prostate glands and seminal vesicles was determined from MR images. T2 signal intensity changes in the seminal vesicles were determined by subtracting higher echo time (TE) from lower TE T2WI. Following treatments all animals were sacrificed, prostate and seminal vesicles collected, and the tissues prepared for histological staining. All data were expressed as mean ± 1 standard deviation. Two-way or multivariate analysis of variance followed by post-hoc test was applied to determine the significant differences. A p-value of <0.05 was considered significant.ResultsHistological analysis indicated tumor in 100% of control mice, whereas 10% of the treated mice showed tumor in prostate gland. Both MRI and measured prostate weights showed higher volume/weight in control mouse group. MRI showed significantly higher volume of seminal vesicles in control animals and T2 signal intensity changes in seminal vesicles of control mice indicating higher number of tumor foci, which was also proven by histology.ConclusionsIn vivo MRI is helpful in determining the efficacy of chemoprevention of prostate cancer in TRAMP mice.

Heterogeneity of buoyancy in response to light between two buoyant types of cyanobacterium Microcystis

Previous investigations suggested that buoyancy state in response to light differed between individuals within natural populations of cyanobacteria. To understand the mechanisms of heterogeneity of buoyancy in different species/strains in relation to light, two types of colonial Microcystis in different buoyancy behavior were selected and used to compare their photosynthetic activity, gas vesicle volume, ballast mass, and migration at varying light regime. The photosynthesis-irradiance curve and F (v)/F (m) examination indicated that negatively buoyant strains were more adapted at high irradiance than buoyant ones. Transcription levels of gvp gene and gas vesicle volume decreased in buoyant strains, but increased in negatively buoyant ones at high irradiance. The results indicated that the combination effect of decrease in gas vesicle buoyancy and increase in carbohydrate contributed to the downward migration of buoyant strains, while the significant increase of gas vesicles provided sufficient buoyancy to negate ballast mass, resulting in the upward migration of negatively buoyant ones at high irradiance. In addition, either sinking or floating velocities were elevated in buoyant and negatively buoyant strains, coincidently with the colony enlargement of all strains at high irradiance, respectively. These findings suggest that this heterogeneity was associated with the interplay between gas vesicles, ballast, and colony size. The fact that different species/strains of Microcystis respond diversely to light depending on their physiological conditions presents a good example to understand heterogeneity of buoyancy in the field, and the presence of heterogeneity of buoyancy may be implicated in the dominancy and persistence of Microcystis bloom in ever-changing environment.

Quantitative textural analysis of Vulcanian pyroclasts (Montserrat) using multi-scale X-ray computed microtomography: comparison with results from 2D image analysis

X-ray computed microtomography (μCT) was carried out on four pyroclasts from the 1997 Vulcanian explosions of Soufrière Hills Volcano, Montserrat. Three-dimensional data from multiple image stacks with different spatial resolutions (0.37, 4–8, and 17.4 μm px−1) were combined to generate size distributions of vesicles, inter-vesicle throats, crystals, and Fe–Ti oxides over a 3.4–860-μm size range, and to compare the results with those obtained by 2D image analysis on the same samples. Qualitative textural observations are in good agreement with those made in 2D, but μCT provides better resolution of textural features and spatial relationships. Calculation of size distributions requires automated decoalescence of the connected vesicle network. Problems related to this process, in part due to the high porosity of pumice, result in potential artefacts in the calculated size distributions, which are discussed in detail. The main modes of the 3D vesicle volume distributions are systematically shifted to larger sizes compared with those of the 2D distributions. Sample total vesicularities obtained in 3D are within 13 vol.% of those found in 2D, and within 10 vol.% of those measured by He-pycnometry. Total number densities of vesicles and Fe–Ti oxides from the two methods are consistent only to the first order, 3D values ranging from 37% to 309% of those in 2D. Vesicle coalescence, investigated by examining inter-vesicle throat size distributions, occurred in all pyroclasts between neighbouring vesicles of many sizes. The larger the vesicle, the more connected it is.

Vesicles with multiple membrane domains

Vesicles with two types of intramembrane domains, both of which are in the fluid state, are studied theoretically using free energy minimization and Monte Carlo simulations. Specific examples are provided by liquid-ordered and liquid-disorderd domains, which are formed by phase separation within multi-component membranes. Multi-domain morphologies, which arise from the interplay between the bending energies of the domains and the line energy of the domain boundaries, are found for a wide range of these elastic parameters and can be further stabilized by the constraint of constant volume, by spontaneous curvatures, and/or by a difference in the Gaussian curvature moduli of the two types of domains. In the latter case, the vesicles attain stable multi-domain morphologies with up to six liquid-disordered domains. Morphologies with three and four such domains have also been observed experimentally, in qualitative agreement with our simulations. For sufficiently small line tension, “lipid rafts” with multiple liquid-ordered domains are also found to be stable if the liquid-ordered domains have a positive spontaneous curvature. The vesicles are found to undergo a variety of transitions between different multi-domain morphologies. Presumably the simplest way to explore these morphological transitions experimentally is by changing the vesicle volume via osmotic deflation.

α‐Chymotrypsin‐Catalyzed Reaction Confined in Block‐Copolymer Vesicles

Herein the reactivity of the enzyme α-chymotrypsin in the confinement of polystyrene-block-poly(acrylic acid) (PS-b-PAA) vesicles was investigated. Enzyme and substrate molecules were encapsulated in PS-b-PAA vesicles with internal diameters ranging from 26 nm to 165 nm during the formation of the vesicles. While the loading efficiencies of enzyme and substrate molecules were practically identical for vesicles of identical size, they were found to increase with decreasing vesicle size. The kinetics of the α-chymotrypsin catalyzed hydrolysis of N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) was evaluated following the increase of the absorption of the product 7-amino-4-methylcoumarin by UV/Vis spectroscopy. The values of the catalytic turnover number obtained for reactions inside vesicles with different sizes showed an increase of up to fourteen times compared to the bulk value with decreasing vesicle volume, while the values of the Michaelis-Menten constant decreased, respectively. This increase in reactivity of α-chymotrypsin is attributed to the effect of vesicle-wall interactions in the finite encapsulated space, where the reagents could diffuse, leading to enhanced collision frequencies.

Spontaneous Protein Crowding in Liposomes: A New Vista for the Origin of Cellular Metabolism

Spontaneous Protein Crowding in Liposomes: A New Vista for the Origin of Cellular Metabolism

Prostate and seminal vesicle volume based consideration of prostate cancer patients for treatment with 3D‐conformal or intensity‐modulated radiation therapya)

The purpose of this article was to determine the suitability of the prostate and seminal vesicle volumes as factors to consider patients for treatment with image-guided 3D-conformal radiation therapy (3D-CRT) or intensity-modulated radiation therapy (IMRT), using common dosimetry parameters as comparison tools. Dosimetry of 3D and IMRT plans for 48 patients was compared. Volumes of prostate, SV, rectum, and bladder, and prescriptions were the same for both plans. For both 3D and IMRT plans, expansion margins to prostate+SV (CTV) and prostate were 0.5 cm posterior and superior and 1 cm in other dimensions to create PTV and CDPTV, respectively. Six-field 3D plans were prepared retrospectively. For 3D plans, an additional 0.5 cm margin was added to PTV and CDPTV. Prescription for both 3D and IMRT plans was the same: 45 Gy to CTV followed by a 36 Gy boost to prostate. Dosimetry parameters common to 3D and IMRT plans were used for comparison: Mean doses to prostate, CDPTV, SV, rectum, bladder, and femurs; percent volume of rectum and bladder receiving 30 (V30), 50 (V50), and 70 Gy (V70), dose to 30% of rectum and bladder, minimum and maximum point dose to CDPTV, and prescription dose covering 95% of CDPTV (D95). When the data for all patients were combined, mean dose to prostate and CDPTV was higher with 3D than IMRT plans (P < 0.01). Mean D95 to CDPTV was the same for 3D and IMRT plans (P > 0.2). On average, among all cases, the minimum point dose was less for 3D-CRT plans and the maximum point dose was greater for 3D-CRT than for IMRT (P < 0.01). Mean dose to 30%, rectum with 3D and IMRT plans was comparable (P > 0.1). V30 was less (P < 0.01), V50 was the same (P > 0.2), and V70 was more (P < 0.01) for rectum with 3D than IMRT plans. Mean dose to bladder was less with 3D than IMRT plans (P < 0.01). V30 for bladder with 3D plans was less than that of IMRT plans (P < 0.01). V50 and V70 for 3D plans were the same for 3D and IMRT plans (P > 0.2). Mean dose to femurs was more with 3D than IMRT plans (P < 0.01). For a given patient, mean dose and dose to 30% rectum and bladder were less with 3D than IMRT plans for prostate or prostate+SV volumes <65 (38/48) and 85 cm3 (39/48), respectively (P < 0.01). The larger the dose to rectum or bladder with 3D plans, the larger also was the dose to these structures with IMRT (P < 0.001). For both 3D and IMRT plans, dose to rectum and bladder increased with the increase in the volumes of prostate and seminal vesicles (P < 0.02 to 0.001). Volumes of prostate and seminal vesicles provide a reproducible and consistent basis for considering patients for treatment with image-guided 3D or IMRT plans. Patients with prostate and prostate+SV volumes <65 and 85 cm3, respectively, would be suitable for 3D-CRT. Patients with prostate and prostate+SV volumes >65 and 85 cm3, respectively, might get benefit from IMRT.

Poroelastic Consolidation in the Phase Separation of Vesicle−Polymer Suspensions

The addition of the polymer poly(diallyl dimethyl ammonium chloride) (polyDADMAC) to multilamellar vesicle dispersions of ditallowethylester dimethyl ammonium chloride (DDAC) causes the vesicle particles to aggregate and form a space-spanning network. These networks collapse under their own buoyant weight. The time evolution of the vesicle network height is investigated as a function of polymer concentration, vesicle volume fraction, and initial sample height. A poroelastic consolidation model accurately relates the time scales of the network collapse to the balance of gravitational, viscous, and elastic forces. Network formation is consistent with an arrested spinodal decomposition of the colloid−polymer mixture; however, while such networks usually lead to nonequilibrium gels, the buoyant stresses are sufficient to drive the phase separation of this colloid−polymer mixture to completion.

Abstract 950: Energy deprivation by Inositol Hexaphosphate inhibits prostate tumor growth and progression in TRAMP mice

Abstract Prostate cancer (PCa) is the second leading cause of cancer-associated deaths in elderly males in Western world, suggesting that newer approaches are needed to prevent/control PCa. Herein, for the first time, by employing anatomical and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), we evaluated non-invasively, the in vivo chemopreventive efficacy of inositol hexaphosphate (IP6), a major constituent of high fiber diets, against prostate tumor growth and progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The study was conducted on a longitudinal basis to simultaneously assess both prostate sizes and changes in tumor vascularization (perfusion and permeability) due to IP6 feeding. Male TRAMP mice, beginning 4 weeks of age, were fed with 1, 2 or 4% (w/v) IP6 in drinking water or only drinking water till 28 weeks of age and monitored using MRI over the course of treatment. Longitudinal assessment of volumes of prostate and seminal vesicles showed profound effect of 2% and 4% IP6 doses on selectively reducing prostate volume without noticeable changes in seminal vesicles. Further, gadolinium-based DCE-MRI showed decreased tumor perfusion and permeability, indicative of anti-angiogenic effects of IP6 treatment. Histopathological analysis at the end of the study corroborated the MRI results, and showed that IP6-fed mice had less severe prostatic lesions compared to positive controls. Specifically, the mice in IP6-fed groups showed higher incidence of prostatic intraepithelial neoplasia (PIN) at the expense of a strong decrease in adenocarcinoma incidence. Importantly, the mice in IP6-fed groups also had prostatic regions with typical normal histology, which was totally absent in positive controls. IHC and WB results indicated anti-proliferative, pro-apoptotic, and anti-angiogenic effect of IP6 feeding, as evidenced by decreased number of PCNA-, PECAM-1/CD-31-, VEGF- and iNOS- positive cells, together with increased number of TUNEL positive cells. As potential mechanisms of IP6 efficacy, decrease in the expression of glucose transporter (GLUT) proteins together with an increase in phosphorylated levels of AMP-activated kinase (AMPKTh172) and acetyl-CoA carboxylase (ACCSer79) were observed in the prostatic tissue of mice from IP6 fed-groups. Since phosphorylation results in decreased activity of ACC which is an essential enzyme for fatty acid synthesis, our findings suggest that IP6 is interfering with the metabolic events occurring in TRAMP prostate tumor tissue. Together, these findings show that oral IP6 feeding blocks PCa growth and progression in TRAMP mouse model via modulation of metabolic events involved in tumor sustenance and thereby results in energy deprivation within the tumor, suggesting its practical and translational potential in suppressing growth and progression of PCa in humans (NCI RO1 grant CA116636). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 950.

A Live-Cell Fluorescence Microplate Assay Suitable for Monitoring Vacuolation Arising from Drug or Toxic Agent Treatment

Lysosomes are membrane-bound subcellular organelles involved in the degradation of macromolecules and pathogens in diverse processes, including endocytosis, phagocytosis, and autophagy. A red fluorescent probe was developed that is selectively sequestered in acidic organelles. U20S cells pretreated with 64 microM chloroquine for as little as 5 h show a dramatic increase in lysosome-like vesicle number and volume. The probe can be employed for highlighting lysosome-like organelles under conditions wherein cells produce vacuoles that contain most of the degradative enzymes of the lysosome but are not as acidic as the parent organelle. Using a conventional fluorescence microplate reader, the half-maximal effective concentration (EC(50)) of chloroquine was estimated. The high Z' score obtained using the assay demonstrated excellent signal-to-noise ratios. The fluorescence microplate assay was successfully employed to screen a small-molecule compound library for agents that increase lysosomal volume and number. One potential application of the new assay is in the toxicology portion of preclinical drug safety assessment (ADME-Tox) workflows, using in vitro cell culture models to aid in the drug development process.