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PubMed2006

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.).

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 microg L(-1)) for 2, 4, 8, 16 and 24h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC(50) of microcystins in carp hepatocytes was 169.2 microg L(-1). The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 microg L(-1)) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 microg L(-1)) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 microg L(-1)), they died in the form of necrosis.

Animals↗
PubMed2006

Role of vinculin in regulating focal adhesion turnover.

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.

Animals↗
PubMed2005

Optical imaging of epileptiform activity in experimentally induced cortical malformations.

Electrophysiological studies of human cortical dysplasia and rodent models revealed widespread hyperexcitability in the malformation itself as well as in its vicinity. We here analyzed the initiation of paroxysmal epileptiform activity using optical imaging of neuronal activity in rats with cortical malformations induced by neonatal freeze lesions. Brain slice preparations were incubated with the voltage-sensitive dye RH795 and neuronal activity was monitored using a fast-imaging photodiode array combined with standard field potential recordings. Spontaneous paroxysmal epileptiform activity emerged in all slices from animals with cortical malformations and sham-operated controls 20-40 min after omission of extracellular Mg(2+). Following electrophysiological and optical recordings, slices were histochemically processed. Using this approach, the present study demonstrated that in animals with freeze-lesion-induced focal cortical malformations, paroxysmal epileptiform activity always emerged from the dysplastic cortex and then spread to adjacent areas through superficial layers. This distribution of initiation sites was significantly different to sham-operated controls in which epileptogenic foci were located in various cytoarchitectonic areas. The present study indicates that following global changes in excitability, the dysplastic cortex itself is the main initiation site of paroxysmal epileptiform activity in animals with focal cortical malformations.

Animals↗
PubMed2006

Entamoeba histolytica: comparison of the role of receptors and filamentous actin among various endocytic processes.

Entamoeba histolytica is the causative agent of amoebic dysentery. Uptake of iron is critical for E. histolytica growth and iron-bound human transferrin (holo-transferrin) has been shown to serve as an iron source in vitro. Although a transferrin-binding protein has been identified in E. histolytica, the mechanism by which this iron source is taken up by this pathogen is not well understood. To gain insight into this process, the uptake of fluorescent-dextran, -holo-transferrin, and human red blood cells (hRBCs) was compared. Both dextran and transferrin were taken up in an apparent receptor-independent fashion as compared to hRBCs, which were taken up in a receptor-mediated fashion. Interestingly, the uptake of FITC-dextran and FITC-holo-transferrin differentially relied on an intact actin cytoskeleton suggesting that their internalization routes may be regulated independently.

Actins↗
PubMed2006

Miltefosine induces apoptosis in arsenite-resistant Leishmania donovani promastigotes through mitochondrial dysfunction.

The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leishmanial chemotherapy is emergence of drug resistance. Miltefosine, the first orally administrable anti-leishmanial drug, has shown the potential against drug-resistant strains of Leishmania. However, there are discrepancies regarding the involvement of P-glycoprotein (Pgp) and sensitivity of miltefosine in multiple drug-resistant (MDR) cell lines that overexpress Pgp in Leishmania. To address this, the effect of miltefosine in arsenite-resistant Leishmania donovani (Ld-As20) promastigotes displaying an MDR phenotype and overexpressing Pgp-like protein was investigated in the current study. Results indicate that Ld-As20 is sensitive to miltefosine. Miltefosine induces process of programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Activation of cellular proteases resulted in activation of DNase that damaged kinetoplast DNA and induced dyskinetoplasty. These data indicate that miltefosine causes apoptosis-like death in arsenite-resistant L. donovani.

Animals↗
PubMed2004

Quantification of substratum contact required for initiation of Colletotrichum graminicola appressoria.

Colletotrichum graminicola, like many plant pathogenic fungi develop appressoria on germling apices, to facilitate penetration of their host. Induction of these structures occurs after contact with the host surface has been established by the germling. Surface contact and subsequent development of appressoria by germlings of C. graminicola was assessed using interference-reflection microscopy (IRM) and microfabricated pillared silicon substrata. Observations with IRM revealed that under low nutrient conditions, 90% of the germlings developed appressoria once they established 4.5 microm of continuous contact with the substratum. Substrata bearing pillars < or =5 microm in width supported < or =10% appressoria; however, as pillar width was increased the percentage of appressoria formed increased in a sigmoid fashion to a maximum of 80%. The percentage of appressoria produced experimentally on these surfaces was compared to data sets generated from a model designed to calculate the probability of appressorium development on similar pillar arrays at various germ tube contact lengths. These results indicate that germ tubes of C. graminicola require more than 4microm of continuous contact with a hydrophobic substratum for induction of appressoria.

Cell Wall↗
PubMed2005

Peroxide resistance in human and mouse lens epithelial cell lines is related to long-term changes in cell biology and architecture.

It is well established that the response of the cell to environmental stress is a major basis for cell modification. Such modification is believed to adapt the cell to better survive its environment. Oxidative stress, a major and ubiquitous stressing factor, was selected for investigating the cellular response to stress. Most studies investigating such cellular response have employed examination of the cell either during or shortly after exposure to stress. We have employed a different approach arguing that the short-term response to stress obscures the biological changes that allow the cell to continue to thrive in its new environment. Reflecting this concept, murine and human cell lines capable of surviving regular exposure to toxic levels of H(2)O(2) or TBOOH have been developed. It was found that certain fundamental long-term changes in cell biology had occurred. The peroxide-resistant cells are diploid rather than aneuploid, show fundamental changes in the cytoskeletal cellular structure, suggesting less rigid more flexible cells, express a new lower molecular mass of p53, a key stress protein responder involved in adaptation, and finally have an immunochemical modification in alphaA-crystallin, a small heat-shock protein. Previously, it was found that there is a dramatic increase in catalase and gluthathione S-transferase activity and a remarkable limited change in expression in other antioxidative genes in these cells. The impact of these changes is discussed. It is apparent that evolutionary cell modifications can occur in response to relatively rapid changes in environment over periods ranging from days to months rather than the thousands of years considered in most evolutionary modifications.

Animals↗
PubMed2005

A novel viscoelastic system from a cationic surfactant and a hydrophobic counterion.

The phase behavior of 2-hydroxy-1-naphthoic acid (2,1-HNC) mixed with cetyltrimethylammonium hydroxide (CTAOH) is reported. This novel system is compared with the published one of 3-hydroxy-2-naphthoic acid (3,2-HNC) mixed with CTAOH. We investigated the phase behavior and properties of the phases in aqueous solutions of 100 mM CTAOH with 2,1-HNC. In both systems a multilamellar vesicle phase is formed when the naphthoate/surfactant ratio (r) reaches unity. When an increasing amount of 2,1-HNC is mixed with a micellar solution of 100 mM CTAOH, an isotropic low-viscous micellar solution, a viscoelastic gel (consisting of rodlike micelles), a turbid region (two-phase region), and a viscoelastic liquid crystalline gel (consisting of multilamellar vesicles, MLV) were formed. The vesicular phase is highly viscoelastic and has a yield stress value. The transition from the micellar to the vesicle phase occurs for CTAOH/2,1-HNC over a two-phase region, where micelles and vesicles coexist. Also it was noticed that 2,1-HNC is dissolved in 100 mM CTAOH until the naphthoate/surfactant ratio reaches approximately 1.5, and the liquid crystalline phases were found to change their color systematically when they were viewed between two crossed polarizers. The vesicles have been characterized by differential interference contrast microscopy, freeze-fracture electron microscopy, and cryo-electron microscopy (cryo-TEM). The vesicles were polydisperse and their diameter ranged from 100 to 1000 nm. The interlamellar spacing between the bilayers was determined with small angle neutron scattering and agrees with the results from different microscopical methods. The complex viscosity rises by six orders of magnitude when rodlike micelles are formed. The complex viscosity decreases again in the turbid region, and then rises approximately six orders of magnitude above the water viscosity. This second rising is due to the formation of the liquid crystalline MLV phase.

Journal Article↗
PubMed2003

The influence of drug incorporation on the structure and release properties of solid dispersions in lipid matrices.

The effect of incorporating caffeine and paracetamol on the structure and behaviour of Gelucire 50/13 has been studied with a view to establishing whether the choice of drug influences the solid structure and release mechanism. Dispersions containing up to 30% w/w drug were prepared and studied using differential scanning calorimetry (DSC), hot stage differential interference contrast microscopy (HSM), dissolution studies and erosion, water uptake (WU) and diameter change measurements. Gelucire 50/13 alone showed a broad melting endotherm using DSC, with two dominant peaks at 36 and 44 degrees C. While incorporation of caffeine did not result in marked changes to the profile, the presence of paracetamol increased the proportion of material in the lower melting peak. HSM studies indicated that the Gelucire crystallised into two main spherulitic conformations; paracetamol appeared to act as a nucleation site for the lower melting fractions while caffeine particles changed into a needle-shaped morphology on cooling the system from the liquid state. Dissolution studies at 37 degrees C showed the caffeine to be released at a relatively faster rate than the paracetamol. Kinetic modeling and direct measurement of the erosion profile indicated that the caffeine systems showed a greater preponderance for erosion than did the corresponding paracetamol systems. It is suggested that the paracetamol promotes the generation of the lower melting form of Gelucire 50/13 which in turn influences the release rate and mechanism. The study therefore indicates that the influence of the drug should be carefully considered when studying Gelucire matrix systems.

Fats↗
PubMed2006

Electronic speckle pattern interferometry: a novel non-invasive tool for studying drug transport rate through free films.

In this work, Electronic Speckle Pattern Interferometry (ESPI) is presented as a non-invasive tool to study drug transport in controlled release systems. ESPI is shown to be a feasible tool to measure drug film permeability via comparison with an ordinary diaphragm cell. A specially designed cuvette was used in the release study: the polymeric film separated the donor and the receiving chambers of the cuvette to create a diffusion cell with no mixing in the two chambers. Thus, the cuvette mimicked a coated system immersed in a stagnant bulk liquid. Concentration profile data were obtained for the two compartments. Using these data, it was possible to visually discriminate between a film subject only to diffusion and a film subject to diffusion as well as osmotic effects. Moreover, using the concentration profile data collected at different time intervals, it was possible to follow the film properties in terms of drug permeability, thus studying how drug permeability depended on drug concentration. Compared to other measuring techniques, ESPI offers the advantages that no invasive measurements are needed, and that no sampling and calibration are required. Furthermore, the permeability can be measured with no influence of mass transfer in the boundary layers.

Benzyl Alcohols↗
PubMed2004

Mechanisms of homogeneous nucleation of polymers of sickle cell anemia hemoglobin in deoxy state.

The primary pathogenic event of sickle cell anemia is the polymerization of the mutant hemoglobin (Hb) S within the red blood cells, occurring when HbS is in deoxy state in the venous circulation. Polymerization is known to start with nucleation of individual polymer fibers, followed by growth and branching via secondary nucleation, yet the mechanisms of nucleation of the primary fibers have never been subjected to dedicated tests. We implement a technique for direct determination of rates and induction times of primary nucleation of HbS fibers, based on detection of emerging HbS polymers using optical differential interference contrast microscopy after laser photolysis of CO-HbS. We show that: (i). nucleation throughout these determinations occurs homogeneously and not on foreign substrates; (ii). individual nucleation events are independent of each other; (iii). the nucleation rates are of the order of 10(6)-10(8)cm(-3)s(-1); (iv). nucleation induction times agree with an a priori prediction based on Zeldovich's theory; (v). in the probed parameter space, the nucleus contains 11 or 12 molecules. The nucleation rate values are comparable to those leading to erythrocyte sickling in vivo and suggest that the mechanisms deduced from in vitro experiments might provide physiologically relevant insights. While the statistics and dynamics of nucleation suggest mechanisms akin to those for small-molecule and protein crystals, the nucleation rate values are nine to ten orders of magnitude higher than those known for protein crystals. These high values cannot be rationalized within the current understanding of the nucleation processes.

Biopolymers↗
PubMed2006

Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter.

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.

Animals↗
PubMed2006

Interaction between cisplatin treated murine peritoneal macrophages and L929 cells: involvement of adhesion molecules, cytoskeletons, upregulation of Ca2+ and nitric oxide dependent cytotoxicity.

Murine peritoneal macrophages on treatment with cisplatin (10 microg/ml) showed increased binding to L929 cells. Cisplatin treated macrophage on co-incubation with L929 cells form a distinct cytoplasmic contact between the two cells. The plasmalemmae of the two cells fuse over a large surface area. The formation of contact between the cisplatin treated macrophage and L929 cell results in the induction of apoptosis in L929 cell. Untreated macrophages did not form a contact with L929 cells and no apoptosis is observed in L929 cells. Immunofluorescence microscopical studies clearly show the participation of cytoskeleton and the adhesion molecules in the formation of contact between the two cells. Further, a significant enhancement of the expression of iNOS and cytosolic Ca2+ was observed in cisplatin treated macrophages co-incubated with L929 cells. Cisplatin treated macrophages produced significant amount of NO when co-incubated with L929 cells, while there was minimal production of NO by untreated macrophages co-incubated with L929 cells. Cisplatin treated macrophage-induced L929 cell death was NO dependent, since L-NMMA (500 microM) significantly inhibited the cytotoxicity of L929 cells. The addition of excess L-arginine (2mM) reversed the L-NMMA induced inhibition of NO production and L929 cell cytotoxicity.

Actin Cytoskeleton↗
PubMed2006

The phylogenetic position of the oxymonad Saccinobaculus based on SSU rRNA.

The oxymonads are a group of structurally complex anaerobic flagellates about which we know very little. They are found in association with complex microbial communities in the guts of animals. There are five recognized families of oxymonads; molecular data have been acquired for four of these. Here, we describe the first molecular data from the last remaining group, represented by Saccinobaculus, an organism that is found exclusively in the hindgut of the wood-eating cockroach Cryptocercus. We sequenced small subunit ribosomal RNA (SSU rRNA) from total gut DNA to describe Saccinobaculus SSU rRNA diversity. We also sequenced SSU rRNA from manually isolated cells of the two most abundant and readily identifiable species: the type species Saccinobaculus ambloaxostylus and the taxonomically contentious Saccinobaculus doroaxostylus. We inferred phylogenetic trees including all five known oxymonad subgroups in order to elucidate the internal phylogeny of this poorly studied group, to resolve some outstanding issues of the taxonomy and identification of certain Saccinobaculus species, and to investigate the evolution of character states within it. Our analysis recovered strong support for the existence of the five subgroups of oxymonads, and consistently united the subgroups containing Monocercomonoides and Streblomastix, but was unable to resolve any further higher-order branching patterns.

Animals↗
PubMed2004

Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons.

Fish embryos exposed to complex mixtures of polycyclic aromatic hydrocarbons (PAHs) from petrogenic sources show a characteristic suite of abnormalities, including cardiac dysfunction, edema, spinal curvature, and reduction in the size of the jaw and other craniofacial structures. To elucidate the toxic mechanisms underlying these different defects, we exposed zebrafish (Danio rerio) embryos to seven non-alkylated PAHs, including five two- to four-ring compounds that are abundant in crude oil and two compounds less abundant in oil but informative for structure-activity relationships. We also analyzed two PAH mixtures that approximate the composition of crude oil at different stages of weathering. Exposure to the three-ring PAHs dibenzothiophene and phenanthrene alone was sufficient to induce the characteristic suite of defects, as was genetic ablation of cardiac function using a cardiac troponin T antisense morpholino oligonucleotide. The primary etiology of defects induced by dibenzothiophene or phenanthrene appears to be direct effects on cardiac conduction, which have secondary consequences for late stages of cardiac morphogenesis, kidney development, neural tube structure, and formation of the craniofacial skeleton. The relative toxicity of the different mixtures was directly proportional to the amount of phenanthrene, or the dibenzothiophene-phenanthrene total in the mixture. Pyrene, a four-ring PAH, induced a different syndrome of anemia, peripheral vascular defects, and neuronal cell death, similar to the effects previously described for potent aryl hydrocarbon receptor ligands. Therefore, different PAH compounds have distinct and specific effects on fish at early life history stages.

Abnormalities, Drug-Induced↗
PubMed2006

Arachidonate 15-lipoxygenase and ubiquitin as fertility markers in boars.

Accurate semen analysis is an important issue in the swine industry. We evaluated two candidate fertility marker proteins associated with sperm cytoplasmic droplet (CD), including 15-lipoxygenase (15-LOX) and ubiquitin (UBI) in a controlled single-sire artificial insemination (AI) trial. Ejaculates (n=116) were collected from 18 fertile Large White boars monthly for 8 mo, and analyzed by semi-quantitative, densitometry-based Western blotting and flow cytometry with antibodies against 15-LOX and UBI. Data were correlated with farrowing rates (FR) and total numbers of piglets born (TNB) from 1754 AI services by 13 of 18 boars, and compared with a conventional microscopic semen analysis. In semi-quantitative Western blotting, both 15-LOX and UBI were correlated with seasonal changes in the percentage of normal (r=-0.38, P<0.01; r=-0.27, P<0.05, respectively) and CD-bearing spermatozoa (r=0.35, P<0.01; r=0.27, P<0.05, respectively). In flow cytometry, UBI and 15-LOX levels showed seasonal changes coinciding with seasonal changes of FR and TNB, representing 13 boars, 88 ejaculates and 1,232 AI services. There were correlations between flow cytometric values of UBI and FR (r=0.31; P<0.05), adjusted FR (r=0.30; P<0.05), TNB (r=-0.38; P<0.01) and adjusted TNB (r=-0.37; P<0.01). Flow cytometric measurements of 15-LOX correlated negatively with TNB (r=-0.33; P<0.05) and adjusted TNB (r=-0.34; P<0.05). These data suggested that boar fertility estimation could be achieved within a group of fertile boars by the use of objectively measurable fertility markers. Flow cytometry appeared more informative and more practical than semi-quantitative Western blotting. This technology could be further optimized for the selection of the most fertile sires in an artificial insemination program.

Animals↗
PubMed2006

The presence of androgen receptors in the epididymis and prostate of the stallion and cryptorchid horse--a preliminary study.

Distribution of androgen receptors (ARs) in the epididymal duct and prostate of three entire stallions and one bilaterally cryptorchid horse was studied immunohistochemically using a polyclonal rabbit antiserum against the ARs. In both the healthy stallions and the cryptorchid, the epithelial cells of the epididymides showed nuclear staining for ARs. The intensity of AR-staining in the principal cells of the epididymis was stronger than that of the basal cells. In the prostate, the glandular secretory cells were moderately stained whereas the basal cells expressed weak AR-staining. Immunostaining for ARs in the reproductive tissues of the cryptorchid horse was always stronger than in those of the stallions. Our results demonstrate for the first time the AR localisation to equine epididymal and prostatic cells, which are directly regulated by androgens.

Animals↗
PubMed2006

Single-stranded DNA of Tomato leaf curl virus accumulates in the cytoplasm of phloem cells.

Geminiviruses have been reported to replicate in, and localize to, the nuclei of host plant cells. We have investigated the tissue and intracellular distribution of the monopartite Tomato leaf curl virus (TLCV) by in situ hybridization. Contrary to the current understanding of geminiviral localization, single-stranded (ss) DNA of TLCV accumulated in the cytoplasm. TLCV ssDNA was also found in the nucleus, as was lower levels of replicative form double-stranded (ds) DNA. Under the same conditions, Tomato golden mosaic virus (TGMV) ssDNA and dsDNA were found in nuclei. ssDNA of TLCV, TGMV, and Tomato yellow leaf curl Sardinia virus (TYLCSV) was detected in some xylem vessels under specific hybridization conditions. Tissue specificity of TLCV was partially released by co-infection with TGMV. Our observations suggest that the mechanism of TLCV movement may differ from that of bipartite begomoviruses.

Cell Nucleus↗