Virus Replication, Cytopathology, and Lysosomal Enzyme Response of Mitotic and Interphase Hep-2 Cells Infected with Poliovirus

Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed.     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.     

Cannabinoid Modulation of Eukaryotic Initiation Factors (eIF2α and eIF2B1) and Behavioral Cross-Sensitization to Cocaine in Adolescent Rats

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Glandular trichomes in Connarus suberosus (Connaraceae): distribution, structural organization and probable functions

Connarus suberosus is a typical species of the Brazilian Cerrado biome, and its inflorescences and young vegetative branches are densely covered by dendritic trichomes. The objective of this study was to report the occurrence of a previously undescribed glandular trichome of this species. The localization, origin and structure of these trichomes were investigated under light, transmission and scanning electron microscopy. Collections were made throughout the year, from five adult specimens of Connarus suberosus near Botucatu, S?o Paulo, Brazil, including vegetative and reproductive apices, leaves and fruits in different developmental stages, as well as floral buds and flowers at anthesis. Glandular trichomes (GTs) occurred on vegetative and reproductive organs during their juvenile stages. The GTs consisted of a uniseriate, multicellular peduncle, whose cells contain phenolic compounds, as well as a multicellular glandular portion that accumulates lipids. The glandular cell has thin wall, dense cytoplasm (with many mitochondria, plastids and dictyosomes), and a large nucleus with a visible nucleolus. The starch present in the plastids was hydrolyzed during the synthesis phase, reducing the density of the plastid stroma. Some plastids were fused to vacuoles, and some evidence suggested the conversion of plastids into vacuoles. During the final activity stages of the GTs, a darkening of the protoplasm was observed in some of the glandular cells, as a programmed cell death; afterwards, became caducous. The GTs in C. suberosus had a temporal restriction, being limited to the juvenile phase of the organs. Their presence on the exposed surfaces of developing organs and the chemical nature of the reserve products, suggest that these structures are food bodies. Field observations and detailed studies of plant-environment interactions, as well as chemical analysis of the reserve compounds, are still necessary to confirm the role of these GTs as feeding rewards.     

Successful crossings with early flowering transgenic poplar: interspecific crossings,but not transgenesis,promoted aberrant phenotypes in offspring

In forest tree species, the reproductive phase is reached only after many years or even decades of juvenile growth. Different early flowering systems based on the genetic transfer of heat‐shock promoter driven flowering‐time genes have been proposed for poplar; however, no fertile flowers were reported until now. Here, we studied flower and pollen development in both HSP::AtFT and wild‐type male poplar in detail and developed an optimized heat treatment protocol to obtain fertile HSP::AtFT flowers. Anthers from HSP::AtFT poplar flowers containing fertile pollen grains showed arrested development in stage 12 instead of reaching phase 13 as do wild‐type flowers. Pollen grains could be isolated under the binocular microscope and were used for intra‐ and interspecific crossings with wild‐type poplar. F1‐seedlings segregating the HSP::AtFT gene construct according to Mendelian laws were obtained. A comparison between intra‐ and interspecific crossings revealed that genetic transformation had no detrimental effects on F1‐seedlings. However, interspecific crossings, a broadly accepted breeding method, produced 47% seedlings with an aberrant phenotype. The early flowering system presented in this study opens new possibilities for accelerating breeding of poplar and other forest tree species. Fast breeding and the selection of transgene‐free plants, once the breeding process is concluded, can represent an attractive alternative even under very restrictive regulations.     

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.     

Plk1 Inhibition Causes Post-Mitotic DNA Damage and Senescence in a Range of Human Tumor Cell Lines

Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.     

Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.