Cerebellar granule cells show age‐dependent migratory differences in vitro

Developmental differences between cerebellar granule cells during their migratory period were revealed using dissociated granule cell cultures isolated from 4, 7, or 10 days old (P4, P7, P10) mice. Under all culture conditions, the great majority of cultivated cell populations consisted of those granule cells that had not reach their final destination in the internal granule cell layer (IGL) by the age of isolation. In vitro morphological development and the expression of migratory markers (TAG‐1, astrotactin, or EphB2) showed similar characteristics between the cultures. The migration of 1008 granule cells isolated from P4, P7, and P10 cerebella and cultivated under identical conditions were analyzed using statistical methods. In vitro time‐lapse videomicroscopy revealed that P4 cells possessed the fastest migratory speed while P10 granule cells retained their migratory activity for the longest time in culture. Cultures obtained from younger postnatal ages showed more random migratory trajectories than P10 cultures. Our observations indicate that despite similar morphological and molecular properties, migratory differences exist in granule cell cultures isolated from different postnatal ages. Therefore, the age of investigation can substantially influence experimental results on the regulation of cell migration. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Changes to the uterine epithelium during the reproductive cycle of two viviparous lizard species (Niveoscincus spp.)

We investigated morphological differences in uterine epithelia of the reproductive cycle between two closely related viviparous skinks, Niveoscincus metallicus (lecithotrophic) and Niveoscincus ocellatus (placentotrophic), which have similar placental complexity but different degrees of placentotrophy. Scanning (SEM) and transmission electron microscopy (TEM) revealed that the uterine surface of non‐reproductive females of both species is mainly covered by ciliated cells. As vitellogenesis begins, the uterine epithelium consists of ciliated and non‐ciliated cells under a thin glycocalyx. Microvilli are greatly reduced at mid‐pregnancy, and the uterus differentiates into two structurally distinct regions: the chorioallantoic and the omphaloplacenta. At late stages of pregnancy, the uterine epithelium of chorioallantoic placenta in both species is further ridged, forming a knobbly uterine surface. The ultrastructural evidence between N. metallicus and N. ocellatus cannot strictly account for the distinct differences in their placentotrophy; as yet unexplored molecular nutrient transport mechanisms that are not reflected in uterine ultrastructure must play significant roles in nutrient transportation. Characteristics consistent with a plasma membrane transformation were confirmed in both species.     

Secondary structure of cell‐penetrating peptides during interaction with fungal cells

Cell‐penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep‐1, MPG, pVEC, TP‐10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α‐helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep‐1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular‐level interaction with the cell membrane, and these simulations suggested that pVEC, TP‐10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α‐helical conformation. In contrast, penetratin, Pep‐1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena.     

Batch‐to‐Batch Variation of Polymeric Photovoltaic Materials: its Origin and Impacts on Charge Carrier Transport and Device Performances

A detailed investigation of the impact of molecular weight distribution of a photoactive polymer, poly[N‐9′‐heptadecanyl‐2,7‐carbazole‐alt‐5,5‐(4′,7′‐di‐2‐thienyl‐2′,1′,3′‐benzothiadiazole)] (PCDTBT), on photovoltaic device performance and carrier transport properties is reported. It is found that different batches of as‐received polymers have substantial differences in their molecular weight distribution. As revealed by gel permeation chromatography (GPC), two peaks can generally be observed. One of the peaks corresponds to a high molecular weight component and the other peak corresponds to a low molecular weight component. Photovoltaic devices fabricated with a higher proportion of low molecular weight component have power conversion efficiencies (PCEs) reduced from 5.7% to 2.5%. The corresponding charge carrier mobility at the short‐circuit region is also significantly reduced from 2.7 × 10^?5 to 1.6 × 10^?8 cm² V^?1 s^?1. The carrier transport properties of the polymers at various temperatures are further analyzed by the Gaussian disorder model (GDM). All polymers have similar energetic disorders. However, they appear to have significant differences in carrier hopping distances. This result provides insight into the origin of the molecular weight effect on carrier transport in polymeric semiconducting materials.     

Over‐expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons

The ability of a yeast cell to propagate [PSI⁺], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI⁺] propagation in dividing cells and they get diluted out as cells multiply. Over‐expression of Hsp104 also leads to [PSI⁺] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over‐expression in [PSI⁺] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion‐free [psi^‐] cells present, no change in the molecular weight of Sup35‐containing SDS‐resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over‐expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over‐expression of Hsp104 causes malpartition of [PSI⁺] propagons between mother and daughter cells in a sub‐population of cells during cell division thereby generating prion‐free [psi^?] cells.     

Influence of the production system on the surface properties of influenza A virus particles

In this study, influenza A/Puerto Rico/8/34 H1N1 virus particles (VP) produced in adherent and suspension Madin Darby canine kidney cells were investigated with a broad analytical toolbox to obtain more information on the VP's surface properties potentially affecting their aggregation behavior. First, differences in aggregation behavior were revealed by VP size distributions obtained via differential centrifugal sedimentation and confirmed by dynamic light scattering. The VP produced in adherent cells showed increased levels of aggregation in a 20 mM NaCl 10 mM Tris‐HCl pH 7.4 low‐salt buffer. This included the formation of multimers (dimers up to pentamers), whereas VP produced in suspension cells displayed no tendency toward aggregate formation. To investigate the cause of these differences in aggregation behavior, the VP samples were compared based on their zeta potential, their surface hydrophobicity, their lipid composition, and the N‐glycosylation of their major VP surface protein hemagglutinin. The zeta potential and the hydrophobicity of the VP produced in the adherent cells was significantly decreased compared to the VP produced in the suspension cells. The lipid composition of both VP systems was approximately identical. The hemagglutinin of the VP produced in adherent cells included more of the larger N‐glycans, whereas the VP produced in suspension cells included more of the smaller N‐glycans. These results indicate that differences in the glycosylation of viral surface proteins should be monitored to characterize VP hydrophobicity and aggregation behavior, and to avoid aggregate formation and product losses in virus purification processes for vaccines and gene therapy.     

AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC‐SIGN

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C‐type lectin dendritic cell‐specific intracellular cell adhesion molecule‐3 (ICAM‐3)‐grabbing non‐integrin (DC‐SIGN), because a detailed characterization at the structural level is lacking. DC‐SIGN recognizes specific Candida‐associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan‐branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope‐based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC‐SIGN. We demonstrate that slight differences in the N‐mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC‐SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 k_BT. The single‐bond affinity of tetrameric DC‐SIGN for wild‐type C. albicans is ~10.7 k_BT and a dissociation constant k_D of 23 μM, which is relatively strong compared with other carbohydrate–protein interactions described in the literature. In conclusion, this study shows that DC‐SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC‐SIGN and its pathogenic ligands will lead to a better understanding of how fungal‐associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti‐fungal drugs. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of distal sugar and interglycosidic linkage of disaccharides on the activity of proline rich antimicrobial glycopeptides

The effect of glycosylation on protein structure and function depends on a variety of intrinsic factors including glycan chain length. We have analyzed the effect of distal sugar and interglycosidic linkage of disaccharides on the properties of proline‐rich antimicrobial glycopeptides, formaecin I and drosocin. Their glycosylated analogs‐bearing lactose, maltose and cellobiose, as a glycan side chain on their conserved threonine residue, were synthesized where these disaccharides possess identical proximal sugar and vary in the nature of distal sugar and/or interglycosidic linkage. The structural and functional properties of these disaccharide‐containing formaecin I and drosocin analogs were compared with their corresponding monoglycosylated forms, β‐d ‐glucosyl‐formaecin I and β‐d ‐glucosyl‐drosocin, respectively. We observed neither major secondary structural alterations studied by circular dichroism nor substantial differences in the toxicity with mammalian cells among all of these analogs. The comparative analyses of antibacterial activities of these analogs of formaecin I and drosocin displayed that β‐d ‐maltosyl‐formaecin I and β‐d ‐maltosyl‐drosocin were more potent than that of respective β‐d ‐Glc‐analog, β‐d ‐cellobiosyl‐analog and β‐d ‐lactosyl‐analog. Despite the differences in their antibacterial activity, all the analogs exhibited comparable binding affinity to DnaK that has been reported as one of the targets for proline‐rich class of antibacterial peptides. The comparative–quantitative internalization studies of differentially active analogs revealed the differences in their uptake into bacterial cells. Our results exhibit that the sugar chain length as well as interglycosidic linkage of disaccharide may influence the antibacterial activity of glycosylated analogs of proline‐rich antimicrobial peptides and the magnitude of variation in antibacterial activity depends on the peptide sequence. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose‐deficient maize cells

As a consequence of the habituation to low levels of dichlobenil (DCB), cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall‐bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB (1.5 μM). Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight‐average relative molecular mass. In addition, polysaccharides underwent massive cross‐linking after being secreted into the cell wall, but this cross‐linking was less pronounced in habituated cells than in non‐habituated ones. However, when relativized, ferulic acid and p‐coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight‐average relative molecular mass, although cross‐linked, as a part of their strategy to compensate for the lack of cellulose.     

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non‐diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.     

Solution structure by nuclear magnetic resonance of the two lantibiotics 97518 and NAI‐107

Lantibiotics 97518 and NAI‐107, produced by the related genera Planomonospora and Microbispora respectively, are members of a family of nisin‐related compounds. They represent promising compounds to treat infections caused by multiresistant Gram‐positive pathogens. Despite their similar structure and a similar antibacterial spectrum, the two lantibiotics exhibit significant differences in their potency. To gain an insight into the structure–activity relationships, their conformational properties in solution are determined by NMR. After carrying out an NOE analysis of 2D ¹H NMR spectra, high‐resolution 3D structures are determined using molecular dynamics simulations. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

WASH‐driven actin polymerization is required for efficient mycobacterial phagosome maturation arrest

Pathogenic mycobacteria survive in phagocytic host cells primarily as a result of their ability to prevent fusion of their vacuole with lysosomes, thereby avoiding a bactericidal environment. The molecular mechanisms to establish and maintain this replication compartment are not well understood. By combining molecular and microscopical approaches we show here that after phagocytosis the actin nucleation‐promoting factor WASH associates and generates F‐actin on the mycobacterial vacuole. Disruption of WASH or depolymerization of F‐actin leads to the accumulation of the proton‐pumping V‐ATPase around the mycobacterial vacuole, its acidification and reduces the viability of intracellular mycobacteria. This effect is observed for M. marinum in the model phagocyte Dictyostelium but also for M. marinum and M. tuberculosis in mammalian phagocytes. This demonstrates an evolutionarily conserved mechanism by which pathogenic mycobacteria subvert the actin‐polymerization activity of WASH to prevent phagosome acidification and maturation, as a prerequisite to generate and maintain a replicative niche.     

A new genus of athecate interstitial dinoflagellates, Togula gen. nov., previously encompassed within Amphidinium sensu lato: Inferred from light and electron microscopy and phylogenetic analyses of partial large subunit ribosomal DNA sequences

The recent emendation of Amphidinium (Dinophyc‐eae), which now only consists of species with minute left‐deflected epicone, has left more than 100 species without a clear generic affiliation. In the present study, a strain identified as one of the species with a divergent epicone type, Amphidinium britannicum (Herdman) Lebour, and six strains resembling A. britannicum but smaller in size were examined by light, scanning and transmission electron microscopy and by sequence analyses of nuclear‐encoded partial large subunit ribosomal DNA to establish their phylog‐eny. Amphidinium britannicum was not closely related to other genera included in the molecular phylogenetic analyses, but formed a highly supported clade in Bayesian analysis together with the six small‐sized strains. The six strains also formed a highly supported clade, consisting of two closely related, albeit distinct, clades. Light and scanning electron microscopy did not reveal significant differences between the vegetative motile cells; however, cells about to undergo mitosis developed longitudinal grooves on the hypocone in one of the clades but not in the other. Both clades differed substantially from A. britannicum in partial large subunit ribosomal DNA as well as in size and shape. Based on morphological similarity and partial large subunit ribosomal DNA evidence, we erect the new genus, Togula gen. nov. with the emended type species Togula britannica (Herdman) comb. nov. Based on differences in division pattern and partial large subunit ribosomal DNA gene divergence we further describe the species Togula compacta (Herdman) comb. nov. and Togula jolla sp. nov.     

Vimentin,colon cancer progression and resistance to butyrate and other HDACis

Dietary fibre protects against colorectal cancer (CRC) most likely through the activity of its fermentation product, butyrate. Butyrate functions as a histone deacetylase inhibitor (HDACi) that hyperactivates Wnt signalling and induces apoptosis of CRC cells. However, individuals who consume a high‐fibre diet may still develop CRC; therefore, butyrate resistance may develop over time. Furthermore, CRC cells that are resistant to butyrate are cross‐resistant to clinically relevant therapeutic HDACis, suggesting that the development of butyrate resistance in vivo can result in HDACi‐resistant CRCs. Butyrate/HDACi‐resistant CRC cells differ from their butyrate/HDACi‐sensitive counterparts in the expression of many genes, including the gene encoding vimentin (VIM) that is usually expressed in normal mesenchymal cells and is involved in cancer metastasis. Interestingly, vimentin is overexpressed in butyrate/HDACi‐resistant CRC cells although Wnt signalling is suppressed in such cells and that VIM is a Wnt activity‐targeted gene. The expression of vimentin in colonic neoplastic cells could be correlated with the stage of neoplastic progression. For example, comparative analyses of LT97 microadenoma cells and SW620 colon carcinoma cells revealed that although vimentin is not detectable in LT97 cells, it is highly expressed in SW620 cells. Based upon these observations, we propose that the differential expression of vimentin contributes to the phenotypic differences between butyrate‐resistant and butyrate‐sensitive CRC cells, as well as to the differences between early‐stage and metastatic colorectal neoplastic cells. We discuss the hypothesis that vimentin is a key factor integrating epithelial to mesenchymal transition, colonic neoplastic progression and resistance to HDACis.     

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014. © 2014 Wiley Periodicals, Inc.     

Differences in the pre‐diapause and pre‐oviposition accumulation of critical nutrients in adult females of the beetle Colaphellus bowringi

In many insect species, the differentiation of development between diapause and reproduction first becomes obvious during the diapause preparation (pre‐diapause) and pre‐oviposition phases. However, the differentiation of nutrient accumulation between these two phases remains unclear. We compared the weights of pre‐diapause and reproductive adult female Colaphellus bowringi Baly (Coleoptera: Chrysomelidae), and measured their triacylglycerol (TAG), protein, and carbohydrate content from emergence until they had fed for 4 days post‐eclosion. We also compared the ovarian development and accumulation of lipid droplets between pre‐diapause and reproductive adult females in order to determine whether we could visually detect differences in nutrient allocation. The weights of both pre‐diapause and reproductive females increased with duration of feeding. The fresh weight and water content of pre‐diapause females was significantly lower than that of reproductive females after feeding for 3 days post‐eclosion. Pre‐diapause females channeled their reserves into TAG in the fat body, whereas reproductive females converted nutrients into proteins and carbohydrates for egg development. These results quantify differences in nutrient accumulation between pre‐diapause and reproductive adult female C. bowringi, and provide clues for understanding the molecular mechanisms underlying differences in the allocation of nutrients between diapause and reproduction in insects.