The fine structure and histochemistry of the caecal epithelium of Calicotyle kröyeri (Monogenea: Monopisthocotylea)

The caecal epithelium of Calicotyle kröyeri consists of a single cell type which functions in the uptake and intracellular digestion of host epidermis and associated mucus. Each cell is columnar with a small basal nucleus and prominent nucleolus. Perinuclear cytoplasm contains narrow profiles of GER and mitochondria with numerous cristae. Golgi complexes are small and indistinct. Most of the cell is filled with vacuoles of heterogeneous content, the largest occupying the cell apex. There is in each cell an apical endocytotic complex comprising cell surface lamellae, apical vesicles and numerous tubular invaginations of the plasmalemma. The limiting membrane of all these components is structurally modified and bears a highly organized array of peg-like structures on its luminal surface. The complex is capable of ingesting particulate food material from the gut lumen for transfer, via vesicles, to the vacuoles for digestion. Most of the vacuoles represent the digestive elements of the cell and, histochemically, are reactive for protein, mucus and carboxylic esterases. Indigestible residues and lipid droplets accumulate in the large apical vacuole and are periodically released to the lumen by exocytosis. Small, undifferentiated caecal cells were occasionally observed in the epithelium, but their development has not been recorded.     

Electron microscopic studies of the lung of the frog

Summary Scanning and transmission electron microscopy were used to study the inner architecture of the frog lung. In some specimens the alveolar surface mucus layer was removed to permit the examination of underlying features. The inner surface of the frog's lung is covered by a layer of microvilli belonging to only one type of epithelial cells. The boundaries of these epithelial cells are demarcated by small ridges. Different degrees of lung expansion cause variations of the surface topography. The morphology of certain surface features is examined in detail. Several methods of drying the specimens are compared.The author wishes to thank Dr. I. E. Richter, Institut für allgemeine und experimentelle Pathologie der Bundeswehr, Mainz, for the opportunity to do these investigations and for helpful discussions.     

Microcystins: measuring human exposure and the impact on human health

Abstract

Context: Freshwater cyanobacterial toxins, microcystins, may be a contributing factor to the development of hepatocellular cancer and colorectal cancer.

Objectives: This review summarizes the toxicity data, exposure routes and the methodologies available to determine exposure to elucidate the relationship to liver and colorectal cancer.

Methods: Literature searches were conducted using Medline, PubMed and Web of Science.

Results: There is evidence of human poisonings resulting from exposure to microcystins, however current methods rely on targeted approaches only suitable for acute exposure. No methods exist for the determination of chronic exposure to microcystins.

Conclusions: With the growing evidence of exposure to microcystins and the possible links to cancer, methods to measure medium to long-term human exposure are needed. The identification and validation of candidate biomarkers are key to undertaking urgently required epidemiological studies.     

Enzymatic esterification of vitamin a in the pigment epithelium of bovine retina

The kinetic properties and subcellular distribution of an esterifying enzyme in the pigment epithelium of bovine retina have been studied using both [1-³H]retinol and [³H]retinol bound to cellular retinol-binding protein as substrates. The most active esterifying fraction in pigment epithelial cell preparations was the microsomes, but the lysosome plus mitochondria fraction also showed some activity, probably due to endoplasmic reticulum present as an impurity. The microsomal enzyme showed optimum activity at pH 7.5, and the reaction was linear up to 30 μg protein and for the first 10–15 min. The apparent K_m values were 16.6 · 10^?6 and 5.5 · 10^?6 M for [³H]retinol and bound [³H]retinol, respectively. This is the first time that retinol bound to cellular retinol-binding protein has been shown to undergo metabolic stransformation. The microsomal esterifying activity was destroyed by boiling for 1 min, or after freezing for 2 months. No clear requirement for ATP, CoA or fatty acid could be demonstrated.Of all the other tissues examined under the same experimental conditions as those used for the pigment epithelium, onlt intestine showed measurable activity. With larger amounts of tissue protein and longer incubation periods, activity was also detectable in microsomes of liver, testis and retina     

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.     

Early oogenesis in the ascidian Ciona savignyi

Summary

Morphology of the germinal epithelium and the early follicular oocyte in the ascidian Ciona savignyi as examined by electron microscopy. The oogenetic part of the germinal epithelium contains oocytes at two different stages and the dark and clear cells. The smaller oocyte contains synaptonemal complexes. The larger oocyte in the initial phase of growth has a conspicuous nucleolus, electron-dense materials and some mitochondria close to the nuclear envelope. The nucleus of the larger oocyte is round and has the smooth contour. The dark cell contains a relatively large nucleus and is sometimes connected to each other by an intercellular bridge. Therefore, the dark cell, which has been suggested to be the progenitor cell of two kinds of accessory cells, may be also the oogonium. The early follicular oocyte just after migration from the germinal epithelium retains most of cytological features similar to those of the larger oocyte. However, the nuclear contour of the early follicular oocyte is uneven. This difference in the nuclear contour probably indicates that such a follicular oocyte is in the second phase of growth.     

Autophagy as the cell survival in response to a microsporidian infection of the midgut epithelium of Isohypsibius granulifer granulifer (Eutardigrada: Hypsibiidae)

The midgut epithelial cells of many invertebrates may possess microorganisms which act as symbionts or pathogens (bacteria, microsporidia, viruses). During our previous studies on Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada, Eutardigrada), which examined alterations of the midgut epithelium during oogenesis, we found that some of the specimens were infected with microsporidia. All stages of pathogens occurred in the cytoplasm of the digestive cells in the midgut epithelium of I. g. granulifer that were infected with microsporidia: meronts, sporonts, sporoblasts, and spores. The cytoplasm of the digestive cells was rich in mitochondria, cisterns of rough endoplasmic reticulum (RER), and Golgi complexes. Autophagy in the digestive cells of the dorsal midgut was much more intensive in comparison with noninfected specimens. Membranes of phagophores surrounded the pathogens forming autophagosomes. These latter structures fused with lysosomes forming autolysosomes and residual bodies appeared. Neither glycogen granules nor droplets of varying electron density, which accumulated in digestive cells during vitellogenesis and choriogenesis, appeared in individuals with microsporidia. While the midgut epithelium in noninfected specimens takes part in vitellogenesis and choriogenesis, in infected specimens, midgut cells are involved in the process of autophagy as a survival strategy.     

Kinase GSK3β functions as a suppressor in colorectal carcinoma through the FTO-mediated MZF1/c-Myc axis

Colorectal carcinoma (CRC) poses heavy burden to human health and has an increasing incidence. Currently, the existing biomarkers for CRC bring about restrained clinical benefits. GSK3β is reported to be a novel therapeutic target for this disease but with undefined molecular mechanisms. Thus, we aimed to investigate the regulatory effect of GSK3β on CRC progression via FTO/MZF1/c-Myc axis. Firstly, the expression patterns of GSK3β, FTO, MZF1 and c-Myc were determined after sample collection. Lowly expressed GSK3β but highly expressed FTO, MZF1 and c-Myc were found in CRC. After transfection of different overexpressed and interference plasmids, the underlying mechanisms concerning GSK3β in CRC cell functions were analysed. Additionally, the effect of GSK3β on FTO protein stability was assessed followed by detection of MZF1 m6A modification and MZF1-FTO interaction. Mechanistically, GSK3β mediated ubiquitination of demethylase FTO to reduce FTO expression. Besides, GSK3β inhibited MZF1 expression by mediating FTO-regulated m6A modification of MZF1 and then decreased the proto-oncogene c-Myc expression, thus hampering CRC cell proliferation. We also carried out in vivo experiment to verify the regulatory effect of GSK3β on CRC via FTO-mediated MZF1/c-Myc axis. It was found that GSK3β inhibited CRC growth in vivo which was reversed by overexpressing c-Myc. Taken together, our findings indicate that GSK3β suppresses the progression of CRC through FTO-regulated MZF1/c-Myc axis, shedding light onto a new possible pathway by which GSK3β regulates CRC.     

A Comprehensive Analysis of Symmetric Arginine Dimethylation in Colorectal Cancer Tissues Using Immunoaffinity Enrichment and Mass Spectrometry

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of methylated peptides combined with high‐resolution mass spectrometry, a total of 147 symmetric dimethyl‐arginine (SDMA) sites in 94 proteins are identified, many of which are RNA binding proteins and enzymes. Quantitative analysis comparing CRC and normal tissues reveals significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of four arginine sites in four proteins. Among the 94 proteins identified in this study, it is confirmed that KH‐type splicing regulatory protein is a target of PRMT5 and highly expressed in CRC tissues compared to noncancerous tissues. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and extends the number of known in vivo SDMA sites. The data obtained are available via ProteomeXchange with the identifier PXD015653.