New Insights in ATP Synthesis as Therapeutic Target in Cancer and Angiogenic Ocular Diseases.

Lactate and ATP formation by aerobic glycolysis, the Warburg effect, is considered a hallmark of cancer. During angiogenesis in non-cancerous tissue, proliferating stalk endothelial cells (ECs) also produce lactate and ATP by aerobic glycolysis. In fact, all proliferating cells, both non-cancer and cancer cells, need lactate for the biosynthesis of building blocks for cell growth and tissue expansion. Moreover, both non-proliferating cancer stem cells in tumors and leader tip ECs during angiogenesis rely on glycolysis for pyruvate production, which is used for ATP synthesis in mitochondria through oxidative phosphorylation (OXPHOS). Therefore, aerobic glycolysis is not a specific hallmark of cancer but rather a hallmark of proliferating cells and limits its utility in cancer therapy. However, local treatment of angiogenic eye conditions with inhibitors of glycolysis may be a safe therapeutic option that warrants experimental investigation. Most types of cells in the eye such as photoreceptors and pericytes use OXPHOS for ATP production, whereas proliferating angiogenic stalk ECs rely on glycolysis for lactate and ATP production. (J Histochem Cytochem XX.XXX-XXX, XXXX).

Diabrotica v. virgifera Seems Not Affected by Entomotoxic Protease Inhibitors from Higher Fungi.

Certain soil insects, such as the root-damaging larvae of the maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), are increasingly difficult to control because of recent bans of some insecticides. An alternative and safer approach may be the development of biopesticides based on entomotoxic defense proteins of higher fungi. Many of these potentially interesting proteins are protease inhibitors, and some have been shown to adversely affect insects. We examined the effects of the cysteine protease inhibitors macrocypin 1, 3, and 4 from Macrolepiota procera, clitocypin from Clitocybe nebularis, and cocaprin 1 and the serine protease inhibitor cospin 1 from Coprinopsis cinerea on D. v. virgifera. We confirmed the inhibition by mycocypins of the cysteine catalytic-type proteolytic activities in gut extracts of larvae and adults. The inhibition of pGlu-Phe-Leu-hydrolyzing activity was stronger than that of Z-Phe-Arg-hydrolyzing activity. Mycocypins and cospin resisted long-term proteolytic digestion, whereas cocaprin 1 was digested. Bioassays with overlaid artificial diet revealed no effects of proteins on neonatal mortality or stunting, and no effects on adult mortality. Immersion of eggs in protein solutions had little effect on egg hatching or mortality of hatching neonates. Microscopic analysis of the peritrophic matrix and apical surface of the midguts revealed the similarity between larvae of D. v. virgifera and the chrysomelid Leptinotarsa decemlineata, which are sensitive to these inhibitors. The resistance of D. v. virgifera to fungal protease inhibitors is likely due to effective adaptation of digestive enzyme expression to dietary protease inhibitors. We continue to study unique protein complexes of higher fungi for the development of new approaches to pest control.

Cell Biology Meets Cell Metabolism: Energy Production Is Similar in Stem Cells and in Cancer Stem Cells in Brain and Bone Marrow.

Energy production by means of ATP synthesis in cancer cells has been investigated frequently as a potential therapeutic target in this century. Both (an)aerobic glycolysis and oxidative phosphorylation (OXPHOS) have been studied. Here, we review recent literature on energy production in glioblastoma stem cells (GSCs) and leukemic stem cells (LSCs) versus their normal counterparts, neural stem cells (NSCs) and hematopoietic stem cells (HSCs), respectively. These two cancer stem cell types were compared because their niches in glioblastoma tumors and in bone marrow are similar. In this study, it became apparent that (1) ATP is produced in NSCs and HSCs by anaerobic glycolysis, whereas fatty acid oxidation (FAO) is essential for their stem cell fate and (2) ATP is produced in GSCs and LSCs by OXPHOS despite the hypoxic conditions in their niches with FAO and amino acids providing its substrate. These metabolic processes appeared to be under tight control of cellular regulation mechanisms which are discussed in depth. However, our conclusion is that systemic therapeutic targeting of ATP production via glycolysis or OXPHOS is not an attractive option because of its unwanted side effects in cancer patients.

Structure, function and development of the digestive system in malacostracan crustaceans and adaptation to different lifestyles.

The digestive system of the malacostracan crustaceans, namely the decapods, isopods, amphipods and mysids, is among the most complex organ systems of the animal kingdom serving multiple functions such as food processing, absorption and storage of nutrients, synthesis of digestive enzymes and blood proteins, detoxification of xenobiotics and osmoregulation. It is rather well investigated compared to other invertebrates because the Malacostraca include many ecological keystone species and food items for humans. The Decapoda and Peracarida share food processing with chewing and filtering structures of the stomach but differ with respect to morphology and ultrastructure of the digestive glands. In the Peracarida, the digestive glands are composed of few, relatively large lateral caeca, whereas in the Decapoda, hundreds to thousands of blindly ending tubules form a voluminous hepatopancreas. Morphogenesis and onset of functionality of the digestive system strongly depend on the mode of development. The digestive system is early developed in species with feeding planktonic larvae and appears late in species with direct lecithotrophic development. Some structures of the digestive system like the stomach ossicles are rather constant in higher taxa and are of taxonomic value, whereas others like the chewing structures are to some degree adapted to the feeding strategy. The nutrient absorbing and storing cells of the digestive glands show considerable ultrastructural variation during moult cycle, vitellogenesis and starvation. Some of the various functions of the digestive system are already assigned to specific sections of the digestive tract and cell types, but others still await precise localization.

Ultrastructural differentiation of plasma membrane and cell junctions in the hindgut cells is synchronized with key developmental transitions in Porcellio scaber.

Differentiation of transporting epithelial cells during development of animal organisms includes remodelling of apical and basal plasma membranes to increase the available surface for transport and formation of occluding junctions, which maintain a paracellular diffusion barrier. This study provides a detailed ultrastructural analysis of apical and basal plasma membrane remodelling and cell junction formation in hindgut cells during late embryonic and early postembryonic development of the crustacean Porcellio scaber. Hindgut cells in late-stage embryos are columnar with flat apical and basal plasma membranes. In early-stage marsupial mancae the hindgut cells begin to acquire their characteristic dome shape, the first apical membrane folding is evident and the septate junctions expand considerably, all changes being probably associated with the onset of active feeding. In postmarsupial mancae the apical labyrinth is further elaborated and the septate junctions are expanded. This coincides with the transition to an external environment and food sources. First basal infoldings appear in the anterior chamber of early-stage marsupial mancae, but in the papillate region they are mostly formed in postmarsupial mancae. In molting late-stage marsupial mancae, the plasma membrane acquires a topology characteristic of cuticle-producing arthropod epithelia and the septate junctions are considerably reduced.

Comparative ultrastructure of cells and cuticle in the anterior chamber and papillate region of Porcellioscaber (Crustacea, Isopoda) hindgut.

Isopod hindgut consists of two anatomical and functional parts, the anterior chamber, and the papillate region. This study provides a detailed ultrastructural comparison of epithelial cells in the anterior chamber and the papillate region with focus on cuticle ultrastructure, apical and basal plasma membrane labyrinths, and cell junctions. Na+/K+-ATPase activity in the hindgut epithelial cells was demonstrated by cytochemical localisation. The main difference in cuticle ultrastructure is in the thickness of epicuticle which is almost as thick as the procuticle in the papillate region and only about one sixth of the thickness of procuticle in the anterior chamber. The apical plasma membrane in both hindgut regions forms an apical plasma membrane labyrinth of cytoplasmic strands and extracellular spaces. In the papillate region the membranous infoldings are deeper and the extracellular spaces are wider. The basal plasma membrane is extensively infolded and associated with numerous mitochondria in the papillate region, while it forms relatively scarce basal infoldings in the anterior chamber. The junctional complex in both hindgut regions consists of adherens and septate junctions. Septate junctions are more extensive in the papillate region. Na+/K+-ATPase was located mostly in the apical plasma membranes in both hindgut regions. The ultrastructural features of hindgut cuticle are discussed in comparison to exoskeletal cuticle and to cuticles of other arthropod transporting epithelia from the perspective of their mechanical properties and permeability. The morphology of apical and basal plasma membranes and localisation of Na+/K+-ATPase are compared with other arthropod-transporting epithelia according to different functions of the anterior chamber and the papillate region.

Cuticle morphogenesis in crustacean embryonic and postembryonic stages.

The crustacean cuticle is a chitin-based extracellular matrix, produced in general by epidermal cells and ectodermally derived epithelial cells of the digestive tract. Cuticle morphogenesis is an integrative part of embryonic and postembryonic development and it was studied in several groups of crustaceans, but mainly with a focus on one selected aspect of morphogenesis. Early studies were focused mainly on in vivo or histological observations of embryonic or larval molt cycles and more recently, some ultrastructural studies of the cuticle differentiation during development were performed. The aim of this paper is to review data on exoskeletal and gut cuticle formation during embryonic and postembryonic development in crustaceans, obtained in different developmental stages of different species and to bring together and discuss different aspects of cuticle morphogenesis, namely data on the morphology, ultrastructure, composition, connections to muscles and molt cycles in relation to cuticle differentiation. Based on the comparative evaluation of microscopic analyses of cuticle in crustacean embryonic and postembryonic stages, common principles of cuticle morphogenesis during development are discussed. Additional studies are suggested to further clarify this topic and to connect the new knowledge to related fields.

Formation of the hindgut cuticular lining during embryonic development of Porcellioscaber (Crustacea, Isopoda).

The hindgut and foregut in terrestrial isopod crustaceans are ectodermal parts of the digestive system and are lined by cuticle, an apical extracellular matrix secreted by epithelial cells. Morphogenesis of the digestive system was reported in previous studies, but differentiation of the gut cuticle was not followed in detail. This study is focused on ultrastructural analyses of hindgut apical matrices and cuticle in selected intramarsupial developmental stages of the terrestrial isopod Porcellioscaber in comparison to adult animals to obtain data on the hindgut cuticular lining differentiation. Our results show that in late embryos of stages 16 and 18 the apical matrix in the hindgut consists of loose material overlaid by a thin intensely ruffled electron dense lamina facing the lumen. The ultrastructural resemblance to the embryonic epidermal matrices described in several arthropods suggests a common principle in chitinous matrix differentiation. The hindgut matrix in the prehatching embryo of stage 19 shows characteristics of the hindgut cuticle, specifically alignment to the apical epithelial surface and a prominent electron dense layer of epicuticle. In the preceding embryonic stage - stage 18 - an electron dense lamina, closely apposed to the apical cell membrane, is evident and is considered as the first epicuticle formation. In marsupial mancae the advanced features of the hindgut cuticle and epithelium are evident: a more prominent epicuticular layer, formation of cuticular spines and an extensive apical labyrinth. In comparison to the hindgut cuticle of adults, the hindgut cuticle of marsupial manca and in particular the electron dense epicuticular layer are much thinner and the difference between cuticle architecture in the anterior chamber and in the papillate region is not yet distinguishable. Differences from the hindgut cuticle in adults imply not fully developed structure and function of the hindgut cuticle in marsupial manca, possibly related also to different environments, as mancae develop in marsupial fluid. Bacteria, evenly distributed within the homogenous electron dense material in the hindgut lumen, were observed only in one specimen of early marsupial manca. The morphological features of gut cuticle renewal are evident in the late marsupial mancae, and are similar to those observed in the exoskeleton.