Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.

Drug abuse, brain calcification and glutamate-induced neurodegeneration

Positive and negative reinforcing systems are part of the mechanism of drug dependence. Drugs with abuse potential may change the manner of response to negative emotional stimuli, activate positive emotional reactions and possess primary reinforcing properties. Catecholaminergic and peptidergic processes are of importance in these mechanisms. Current research needs to understand the types of adaptations that underlie the particularly long-lived aspects of addiction. Presently, glutamate is candidate to play a role in the enduring effects of drugs of abuse. For example, it participates in the chronic pathological changes of corticostriatal terminals produced by methamphetamine. At the synaptic level, a link between over-activation of glutamate receptors, [C(a2+)](i) increase and neuronal damage has been clearly established leading to neurodegeneration. Thus, neurodegeneration can start after an acute over-stimulation whose immediate effects depend on a diversity of calcium-activated mechanisms. If sufficient, the initial insult results in calcification and activation of a chronic on-going process with a progressive loss of neurons. At present, long-term effects of drug dependence underlie an excitotoxicity process linked to a polysynaptic pathway that dynamically regulates synaptic glutamate. Retaliatory mechanisms include energy capability of the neurons, inhibitory systems and cytoplasmic calcium precipitation as part of the neuron-glia interactions. This paper presents an integrated view of these molecular and cellular mechanisms to help understand their relationship and interdependence in a chronic pathological process that suggest new targets for therapeutic intervention.

Spermiogenesis and spermatozoon ultrastructure of the dilepidid cestode Molluscotaenia crassiscolex (von Linstow, 1890), an intestinal parasite of the common shrew Sorex araneus

Spermiogenesis in Molluscotaenia crassiscolex begins with the formation of a differentiation zone containing two centrioles. One of the centrioles develops a flagellum directly into the cytoplasmic extension. The nucleus elongates and later migrates along the spermatid body. During advanced stages of spermiogenesis, a periaxonemal sheath appears in the spermatid. Spermiogenesis finishes with the appearance of a single helicoidal crested body at the base of the spermatid and, finally, the narrowing of the ring of arched membranes causes the detachment of the fully formed spermatozoon. The mature spermatozoon of M. crassiscolex exhibits a partially detached crested body in the anterior region of the spermatozoon, one axoneme, twisted cortical microtubules, a periaxonemal sheath, and a spiralled nucleus. The anterior spermatozoon extremity is characterized by the presence of an electron-dense apical cone and a single spiralled crested body, which is attached to the sperm cell in the anterior and posterior areas of region I, whereas in the middle area it is partially detached from the cell. This crested body is described for the first time in cestodes. The posterior extremity of the male gamete exhibits only the disorganizing axoneme. Results are discussed and compared particularly with the available ultrastructural data on dilepidids sensu lato.

Spermiogenesis and spermatozoon ultrastructure of the diphyllidean cestode Echinobothrium euterpes (Neifar, Tyler and Euzet 2001) Tyler 2006, a parasite of the common guitarfish Rhinobatos rhinobatos

Spermiogenesis and the ultrastructural characters of the spermatozoon of Echinobothrium euterpes are described by means of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a common guitarfish Rhinobatos rhinobatos caught in the Gulf of Gabès (Tunisia). Spermiogenesis in E. euterpes is characterized by the orthogonal development of two unequal flagella followed by the flagellar rotation and the proximodistal fusion of these flagella with the median cytoplasmic process. The most interesting pattern characterizing the diphyllidean cestodes is the presence of a triangular body constituted by fines and dense granules without visible striation and assimilated at the striated rootlets. This pattern, only related in the Diphyllidea cestodes may be a synapomorphy of this order. Spermiogenesis is also characterized by the presence of a very short flagellum (around 1 μm long), observed in all the stages of spermiogenesis. This type of flagellum has never been commented in the diphyllidean cestodes and should be considered as an evolved character in this group. In the latest stage of spermiogenesis, this short axoneme probably degenerates. Thus, the mature spermatozoon of E. euterpes possesses only one axoneme of 9 + '1' trepaxonematan pattern. It also exhibits a single helical electron-dense crested body, a spiraled nucleus, few parallel cortical microtubules, and α-glycogen granules. Similitudes and differences between spermatozoa of diphyllideans are discussed.

Spermiogenesis and spermatozoon ultrastructure of the diphyllidean cestode Echinobothrium euterpes (Neifar, Tyler and Euzet 2001) Tyler 2006, a parasite of the common guitarfish Rhinobatos rhinobatos

Spermiogenesis and the ultrastructural characters of the spermatozoon of Echinobothrium euterpes are described by means of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a common guitarfish Rhinobatos rhinobatos caught in the Gulf of Gabès (Tunisia). Spermiogenesis in E. euterpes is characterized by the orthogonal development of two unequal flagella followed by the flagellar rotation and the proximodistal fusion of these flagella with the median cytoplasmic process. The most interesting pattern characterizing the diphyllidean cestodes is the presence of a triangular body constituted by fines and dense granules without visible striation and assimilated at the striated rootlets. This pattern, only related in the Diphyllidea cestodes may be a synapomorphy of this order. Spermiogenesis is also characterized by the presence of a very short flagellum (around 1 μm long), observed in all the stages of spermiogenesis. This type of flagellum has never been commented in the diphyllidean cestodes and should be considered as an evolved character in this group. In the latest stage of spermiogenesis, this short axoneme probably degenerates. Thus, the mature spermatozoon of E. euterpes possesses only one axoneme of 9 + '1' trepaxonematan pattern. It also exhibits a single helical electron-dense crested body, a spiraled nucleus, few parallel cortical microtubules, and α-glycogen granules. Similitudes and differences between spermatozoa of diphyllideans are discussed.

SMRT-mediated co-shuttling enables export of class IIa HDACs independent of their CaM kinase phosphorylation sites

The Class IIa histone deacetylases (HDAC)4 and HDAC5 play a role in neuronal survival and behavioral adaptation in the CNS. Phosphorylation at 2/3 N-terminal sites promote their nuclear export. We investigated whether non-canonical signaling routes to Class IIa HDAC export exist because of their association with the co-repressor Silencing Mediator Of Retinoic And Thyroid Hormone Receptors (SMRT). We found that, while HDAC5 and HDAC4 mutants lacking their N-terminal phosphorylation sites (HDAC4(MUT), HDAC5(MUT)) are constitutively nuclear, co-expression with SMRT renders them exportable by signals that trigger SMRT export, such as synaptic activity, HDAC inhibition, and Brain Derived Neurotrophic Factor (BDNF) signaling. We found that SMRT's repression domain 3 (RD3) is critical for co-shuttling of HDAC5(MUT), consistent with the role for this domain in Class IIa HDAC association. In the context of BDNF signaling, we found that HDAC5(WT), which was more cytoplasmic than HDAC5(MUT), accumulated in the nucleus after BDNF treatment. However, co-expression of SMRT blocked BDNF-induced HDAC5(WT) import in a RD3-dependent manner. In effect, SMRT-mediated HDAC5(WT) export was opposing the BDNF-induced HDAC5 nuclear accumulation observed in SMRT's absence. Thus, SMRT's presence may render Class IIa HDACs exportable by a wider range of signals than those which simply

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Membrane interaction of a new synthetic antimicrobial lipopetide sp-85 with broad spectrum activity

Antimicrobial peptides offer a new class of therapeutic agents to which bacteria may not be able todevelop genetic resistance, since their main activity is in the lipid component of the bacterial cell mem-brane. We have developed a series of synthetic cationic cyclic lipopeptides based on natural polymyxin,and in this work we explore the interaction of sp-85, an analog that contains a C12 fatty acid at theN-terminus and two residues of arginine. This analog has been selected from its broad spectrum antibac-terial activity in the micromolar range, and it has a disruptive action on the cytoplasmic membrane ofbacteria, as demonstrated by TEM. In order to obtain information on the interaction of this analog withmembrane lipids, we have obtained thermodynamic parameters from mixed monolayers prepared withPOPG and POPE/POPG (molar ratio 6:4), as models of Gram positive and Gram negative bacteria, respec-tively. LangmuirBlodgett films have been extracted on glass plates and observed by confocal microscopy,and images are consistent with a strong destabilizing effect on the membrane organization induced bysp-85. The effect of sp-85 on the membrane is confirmed with unilamelar lipid vesicles of the same com-position, where biophysical experiments based on fluorescence are indicative of membrane fusion andpermeabilization starting at very low concentrations of peptide and only if anionic lipids are present.Overall, results described here provide strong evidence that the mode of action of sp-85 is the alterationof the bacterial membrane permeability barrier.

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.