Katarina Kapuralin

Spectrophotometric analysis of flavonoid-DNA interactions and DNA damaging/protecting and cytotoxic potential of flavonoids in human peripheral blood lymphocytes.

The ability of luteolin, kaempferol and apigenin to bind to calf thymus (ct)-DNA, mode of action and stability of flavonoids in buffer were investigated. Spectrophotometric analysis revealed a rapid degradation of apigenin in an aqueous medium, while kaempferol and luteolin were stable for 24h upon dissolution in water. Spectrophotometric study of the interactions of kaempferol and luteolin with calf thymus DNA suggests classic intercalation as their dominant binding mode to DNA. Cytotoxicity/genotoxicity and cytoprotective/genoprotective effects of flavonoids in non-stressed and hydrogen peroxide stressed human peripheral lymphocytes were investigated using the fluorescent dye exclusion method and alkaline comet assay. Flavonoids revealed significant genoprotective effects in hydrogen peroxide stressed cells and in cells submitted to longer incubation in the cell culture medium. Luteolin, followed by apigenin and kaempferol, was shown to be the most effective in protecting DNA from oxidative damage induced by hydrogen peroxide. However, the investigated flavonoids also induced DNA damage, indicating their prooxidative capacity. The balance between the protection of DNA from oxidative damage and prooxidative effects was strongly dependent on flavonoid concentration and the incubation period.

Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion

Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second‐order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)‐derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3–L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion. Copyright

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.

Krüppel-like transcription factor 8 (Klf8) is expressed and active in the neurons of the mouse brain.

Krüppel-like transcription factor 8 (KLF8) is a transcription factor suggested to be involved in various cellular events, including malignant cell transformation, still its expression in the adult rodent brain remained unknown. To analyze Klf8 in the mouse brain and to identify cell types expressing it, a specific transgenic Klf8(Gt1Gaj) mouse was used. The resulting Klf8 gene-driven β-galactosidase activity was visualized by X-gal histochemical staining of the brain sections. The obtained results were complemented by in situ RNA hybridization and immunohistochemistry. Klf8 was highly expressed throughout the adult mouse brain gray matter including the cerebral cortex, hippocampus, olfactory bulb, hypothalamus, pallidum, and striatum, but not in the cerebellum. Immunofluorescent double-labeling revealed that KLF8-immunoreactive cells were neurons, and the staining was located in their nucleus. This was the first study showing that Klf8 was highly expressed in various regions of the mouse brain and in particular in the neurons, where it was localized in the cell nuclei.

Neurons and a subset of interstitial cells of Cajal in the enteric nervous system highly express Stam2 gene.

Signal transducing adaptor molecule 2 (STAM2) is a phosphotyrosine protein, which is a member of the endosomal sorting complex required for transport (ESCRT‐0) and is involved in the sorting process of the mono‐ubiquitinated endosomal cargo for degradation in the lysosome. Analysis of gene trap mice carrying lacZ in frame with Stam2 revealed beta‐galactosidase activity in the enteric nervous system (both in the myenteric and submucosal plexus) throughout the digestive tract. STAM2 immunostaining confirmed that the observed beta‐galactosidase activity coincided with high Stam2 expression. To identify cell types with high Stam2 expression, STAM2 immunostaining was colocalized with the neuronal markers microtubule‐associated protein 2 and protein gene product 9.5 and with c‐kit as a marker for interstitial cells of Cajal (ICCs). STAM2 and c‐kit positive cells comprised a subset of ICCs in the enteric nervous system. Qualitative and quantitative analysis of the morphology of the enteric nervous system in the homozygous mice carrying gene trap insertion in the Stam2 gene did not reveal phenotype changes; therefore, STAM2 function in the digestive tube remains elusive. Anat Rec, 2012.

Stam2 expression pattern during embryo development.

STAM2 is a tyrosine-phosphorylated protein suggested to be involved in cargo selection during endocytic pathway, regulation of exocytosis and intracellular signaling. Gene trap method was used to create via insertional mutagenesis a mutant mouse line with integration of promoterless βgeo (lacZ-neomycin phosphotransferase fusion) gene in the second intron of Stam2 gene, enabling analysis of its in vivo expression and function. The inserted β-galactosidase (lacZ) reporter gene was used to reveal Stam2 expression during development. Stam2 in situ RNA hybridization and immunostaining confirmed the observed β-galactosidase activity reflecting high Stam2 expression. The homozygous mutant mice showed no overt phenotypic alterations. Stam2 expression was detected after E9.5 in the gut, notochord, neural tube and heart. In the nervous system it was located in the floor, roof and basal plates of the developing neural tube, and in the developing cortex, hippocampus and olfactory bulbs. Toward the end of gestation, Stam2 expression appeared in the testis and ovary, lungs, nasal cavity epithelium, kidneys, urogenital sinus, intestine, pancreas, pituitary and adrenal glands, muscles, brown adipose tissue, skin and epithelium of the tongue and oral cavity.