Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Kiwifruit extracts inhibit cytokine production by lipopolysaccharide-activated macrophages, and intestinal epithelial cells isolated from IL10 gene deficient mice

Inflammatory bowel disease (IBD) is a chronic, inflammatory disorder of the gastrointestinal tract involving an inappropriate immune response to commensal microorganisms in a genetically susceptible host. This study examined the effects of aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (Actinidia deliciosa) using in vitro models of IBD. These models comprised primary macrophages and intestinal epithelial cells isolated from C57BL/5J and interleukin-10 gene deficient (Il10^−/−) mice and RAW 264.7, a murine macrophage-like cell line. All four kiwifruit extracts reduced the activation of these models after lipopolysaccharide stimulation, decreasing nitric oxide and cytokine secretion by both Il10^−/− and wild-type cells. The ethyl acetate extracts exhibited the highest anti-inflammatory activity, with almost complete suppression of lipopolysaccharide-stimulated macrophage activation. These results suggest that kiwifruit extracts have significant anti-inflammatory activity relevant to IBD. We suggest that the Il10^−/− mouse is a suitable model for further study of these compounds.     

A Battery of Cell- and Structure-specific Markers for the Adult Porcine Retina

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathological changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and reproducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina. (J Histochem Cytochem 58:377–389, 2010)     

Plant regeneration via organogenesis from adventitious bud explants of a medicinal herb species, <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis>

Summary Explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet regeneration, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal portion of the plant. The optimum hormone combination for plantlet regenertion was 4.44 μM benzyladenine plus 2.26 μM 2,4-dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6% of the adventitious bud explants, with an average of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medium with 4.57 μM α-naphthaleneacetic acid, as 92% of regenerated buds rooted. Regenerated plantlets were successfully transferred to a greenhouse with 86% survival. Histological observation indicated that new adventitious buds originated from the superficial meristematic cell cluster of the granular callus induced from adventitious bud explants via organogenesis.     

Histology of “placoderm” dermal skeletons: Implications for the nature of the ancestral gnathostome

The vertebrate dermal skeleton has long been interpreted to have evolved from a primitive condition exemplified by chondrichthyans. However, chondrichthyans and osteichthyans evolved from an ancestral gnathostome stem‐lineage in which the dermal skeleton was more extensively developed. To elucidate the histology and skeletal structure of the gnathostome crown‐ancestor we conducted a histological survey of the diversity of the dermal skeleton among the placoderms, a diverse clade or grade of early jawed vertebrates. The dermal skeleton of all placoderms is composed largely of a cancellar architecture of cellular dermal bone, surmounted by dermal tubercles in the most ancestral clades, including antiarchs. Acanthothoracids retain an ancestral condition for the dermal skeleton, and we record its secondary reduction in antiarchs. We also find that mechanisms for remodeling bone and facilitating different growth rates between adjoining plates are widespread throughout the placoderms. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Nicotinic acetylcholine receptors (nAChRs) at zebrafish red and white muscle show different properties during development

Nicotinic acetylcholine receptors (nAChRs) are highly expressed at the vertebrate neuromuscular junction (NMJ) where they are required for muscle activation. Understanding the factors that underlie NMJ development is critical for a full understanding of muscle function. In this study we performed whole cell and outside‐out patch clamp recordings, and single‐cell RT‐qPCR from zebrafish red and white muscle to examine the properties of nAChRs during the first 5 days of development. In red fibers miniature endplate currents (mEPCs) exhibit single exponential time courses at 1.5 days postfertilization (dpf) and double exponential time courses from 2 dpf onwards. In white fibers, mEPCs decay relatively slowly, with a single exponential component at 1.5 dpf. By 2 and 3 dpf, mEPC kinetics speed up, and decay with a double exponential component, and by 4 dpf the exponential decay reverts back to a single component. Single channel recordings confirm the presence of two main conductance classes of nAChRs (～45 pS and ～65 pS) in red fibers with multiple time courses. Two main conductance classes are also present in white fibers (～55 pS and ～73 pS), but they exhibit shorter mean open times by 5 dpf compared with red muscle. RT‐qPCR of mRNA for nicotinic receptor subunits supports a switch from γ to ε subunits in white fibers but not in red. Our findings provide a developmental profile of mEPC properties from red and white fibers in embryonic and larval zebrafish, and reveal previously unknown differences between the NMJs of these muscle fibers.© 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 916–936, 2016     

Histological and morphological evaluations of the digestive tract and associated organs of haddock throughout post-hatching ontogeny

At hatching, the oesophagus of haddock Melanogrammus aeglefinus lacks goblet cells, the intestine is a simple undifferentiated tube, the liver is present as a rounded mass caudal to the heart, and numerous zymogen granules are present in the pancreas. The first intestinal convolution appears at day 2, at the posterior end of the digestive tract. The oesophagus displays alcian blue and PAS positive mucus secreting cells on day 12, which become numerous by day 15. By day 18, epithelial cells of the posterior intestine show evidence of protein absorption in the form of supranuclear vacuoles. The swimbladder inflates in 50% of the larvae by day 22, although inflation rate is highly variable. By day 35, or 10 mm, a pyloric caecal ridge appears which separates the presumptive stomach, which is now showing evidence of gastric gland formation, from the intestine. This marks the beginning of digestive features characteristic of the juvenile stage.     

Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.     

Unexpected microanatomical variation among Eocene Antarctic stem penguins (Aves: Sphenisciformes)

The microanatomical and histological structure of Eocene Antarctic stem penguin tarsometatarsi is examined in order to characterise the bone microstructure. Eight adult tarsometatarsi belonging to eight fossil species (Palaeeudyptes gunnari, Palaeeudyptes klekowskii, Anthropornis grandis, Anthropornis nordenskjoeldi, Archaeospheniscus wimani, Marambiornis exilis, Delphinornis arctowskii and Delphinornis larseni) collected from the Antarctic A. nordenskjoeldi Biozone (La Meseta Formation, ～34.2 Ma) were examined. The thin sections revealed a distinctive microanatomical variation among taxa. Whereas Anthropornis spp., A. wimani and P. gunnari possess massive, clearly osteosclerotic bones (medullary cavities absent or strongly reduced), the bones of Delphinornis spp., P. klekowski and M. exilis exhibit well-developed medullary cavities. The cortical bone in all the specimens consists of primary, well-vascularised fibro-lamellar bone and variable amounts of secondary bone. Medullary cavities are coated by a thick layer of lamellar bone tissue and coarse compacted cancellous bone. Although several causes can explain the striking microanatomical variation (e.g. ontogeny), we interpret that such variation is related to differential adaptations to the aquatic life, for which taxa with more massive bones were possibly adapted to deeper and more prolonged diving excursions.     

Morphological and functional integrity of precision-cut rat liver slices in rotating organ culture and multiwell plate culture: Effects of oxygen tension

We examined the maintenance of functional and morphological integrity of precision-cut rat liver slices cultured in various incubation systems and conditions for 72 h. Slices were incubated (37°C) for 6, 24, 48, and 72 h in supplemented Williams E medium in 6-well plastic culture plates on a gyratory shaking platform (WPCS) or in a rotating organ culture system (ROCS) using 5% CO₂–95% air (WPCS/air or ROCS/air) or 5% CO₂–70% O₂–25% N₂ (WPCS/ O₂ or ROCS/ O₂). Biochemical and functional parameters of slices maintained in WPCS/air or WPCS/ O₂ were almost totally inhibited after 24 h, in keeping with the extensive and diffuse coalescing coagulative necrosis typical of post-ischemic injury affecting almost all the slice surface after 48 h. As compared to freshly isolated slices, slices maintained in ROCS/air for 72 h showed stable ATP and GSH content, increased protein synthesis, and a slight steady decrease in GST activity, while ATP and GST activity remained stable and protein synthesis and GSH content increased in slices incubated in ROCS/ O₂ for 72 h. The extent of coagulative necrosis was markedly lower in longitudinal sections from slices incubated for 72 h in ROCS/ O₂ than in ROCS/air. Transversal sections from slices kept in ROCS/air for 72 h showed a thick central band of necrotic cells edged by two peripheral layers of viable hepatocytes, whereas most of the slice was composed of viable hepatocytes lined by two thin layers of necrotic cells after 72 h in ROCS/ O₂. ROCS/ O₂ emerged as the system best preserving the histological and functional integrity of rat liver slices in long-term culture.