Caveolin-1 regulates human trabecular meshwork cell adhesion,endocytosis, and autophagy

Impaired trabecular meshwork (TM) outflow is implicated in the pathogenesis of primary open-angle glaucoma (POAG). We previously identified the association of a caveolin-1 (CAV1) variant with POAG by genome-wide association study. Here we report a study of CAV1 knockout (KO) effect on human TM cell properties. We generated human CAV1-KO TM cells by CRISPR/Cas9 technology, and we found that the CAV1-KO TM cells less adhered to the surface coating than the wildtype TM cells by 69.34% ( P < 0.05), but showed no difference in apoptosis. Higher endocytosis ability of dextran and transferrin was also observed in the CAV1-KO TM cells (4.37 and 1.89-fold respectively, P < 0.001), compared to the wildtype TM cells. Moreover, the CAV1-KO TM cells had higher expression of extracellular matrix-degrading enzyme genes ( ADMTS13 and MMP14) as well as autophagy-related genes ( ATG7 and BECN1) and protein (LC3B-II) than the wildtype TM cells. In summary, results from this study showed that the CAV1-KO TM cells have reduced adhesion with higher extracellular matrix-degrading enzyme expression, but increased endocytosis and autophagy activities, indicating that CAV1 could be involved in the regulation of adhesion, endocytosis, and autophagy in human TM cells.     

SIMILARITIES IN THE ASEXUAL REPRODUCTION OF THE OCEANIC DINOFLAGELLATES, PYROCYSTIS FUSIFORMIS, PYROCYSTIS LUNULA, AND PYROCYSTIS NOCTILUCA1

Three cultured species of Pyrocystis (Dinoccoccales) reproduced asexually by forming 2 (or 1) aplanospores or zoospores inside the parent cell wall. In all 3 species these small reproductive cells, although they may not resemble the parent cells, swell up rapidly (～ 10 min) to the approximate size and shape of the parent cell. These swollen cells become new vegetative cells. The above asexual process is the only way by which cells numbers increase in our cultures. Pyrocystis lunula was propagated at the lunula stage of the life cycle. The nonmotile crescent-shaped cells produced reproductive cells that were Gymnodinium-shaped and had, in some cases, a trailing flagellum. With P. fusiformis and P. noctiluca, the reproductive cells were not flagellated. With P. fusiformis, these bodies had a pronounced equatorial constriction like a girdle, while in P. noctiluca the “girdle” was an inconspicuous feature if present. With P. noctiluca and P. fusiformis on a 12:12 ld cycle, reproductive cells were formed early in the dark period and they swelled up at the beginning of the light period. Reproduction of P. lunula was not well phased in our experiments, with reproductive cells developing at the end of the light period and the end of the dark period.     

MICA, a new polymorphic HLA-related antigen, is expressed mainly by keratinocytes, endothelial cells, and monocytes

 MICA is a new polymorphic gene in the HLA region expressed in epithelial cell lines and gastrointestinal epithelium. Little is yet known about the MICA protein, and the pattern of its expression by freshly isolated cells has not been established. In the present experiments, we used antibodies raised in rabbits against α1 and α2 domain-peptides to study the expression of MICA. By western blot and immunoprecipitation, we detected a band of 62 000 M _r in various cell lines (THP-1, U937, HeLa, A431, Raji, MOLT-4, and HUV-EC-C) and in freshly isolated keratinocytes, endothelial cells, and monocytes but not in CD4⁺ and CD8⁺ T cells, and CD19⁺ cells (B lymphocytes). It was not possible to up-regulate the expression of MICA in different cells by stimulation with γ-interferon, but the expression of MICA was induced in phytohemagglutinin-stimulated T cells. We confirmed that MICA is expressed at the cell surface by flow cytometry. Results of immunoprecipitation studies of β₂-microglobulin (β₂m)- or MICA-depleted, metabolically labeled HeLa cells indicated that MICA was not associated with β₂m. Although the function of MICA is still unknown, its restricted pattern of tissue expression, the fact that it is expressed on the cell surface, and its polymorphic nature suggest that this new molecule, encoded close to HLA class I, may play a role in the interaction between epithelial cells and cells of the immune system. Received: 21 May 1997 / Revised: 15 July 1997     

CONIDIOGENESIS IN CERATOCYSTIS ULMI,CERATOCYSTIS PICEAE,AND GRAPHIUM PENICILLIOIDES

Conidiogenous cells of both the synnematous and mononematous conidiophores in Ceratocystis ulmi and C. piceae develop by sympodial proliferation. Holoblastic conidia are produced on nodules or short denticles from the synnematous conidiogenous cells and on well-defined denticles from the mononematous conidiogenous cells. Graphium penicillioides is characterized by percurrent proliferation of the conidiogenous cells and the conidia are holoblastic and annellidic. A comparison of the type material of G. penicillioides with the lectotype specimen of C. piceae indicates that G. penicillioides is not the conidial state of C. piceae. The method of conidial development in C. ulmi and C. piceae is distinct from that of G. penicillioides, the lectotype species of Graphium; these conidial states are, therefore, placed in a new hyphomycete genus, Pesotum, as the Pesotum state of Ceratocystis ulmi and C. piceae, respectively.     

Adhesion,proliferation, and adipogenesis in primary rat cell cultures: effects of collagenous substrata,fibronectin, and serum

Summary The effects of collagenous substrata, fibronectin, and fetal bovine serum on the adhesion, proliferation, and adipogenesis of rat stromal-vascular cells are reported. There was no effect on initial stromal-vascular cell-attachment by fetal bovine serum or fibronectin. The number of cells attached to a hydrated collagen-gel was almost twice (P<0.04) the number attached to dried collagen-gel or dried denatured collagen-gel. Total number of cells after 5 days in culture was similar among the collagenous substrata and among the treatments with or without fibronectin in the growth media. Total number of cells increased significantly (P<0.02) with 10% FBS. Adipocytic formation was inhibited by hydrated collagen-gel (P<0.02) compared to dried collagen-gel or dried, denatured collagenous substrata. An interaction occurred between dried, denatured gel and fetal bovine serum so that total formation of adipocytes increased by increasing the level of fetal bovine serum (P<0.07). Adipocytic formation was inhibited by hydrated collagen-gel at all levels of fetal bovine serum. The percentage of cells that converted to adipocytes was significantly lower (P<0.01) on hydrated collagen-gel compared to dried, denatured or dried collagen-gel. Percentage of conversion was not significantly different among levels of fetal bovine serum, although this percentage increased as fetal bovine serum level increased. Adipocytic conversion was not different between fibronectin-treated or untreated cells. Morphology of stromal vascular cells was similar on dried collagen and dried, denatured collagen-gel, but tended to remain bipolar on hydrated collagen-gel. These studies indicate that fetal bovine serum in combination with the extracellular matrix (dried, denatured collagen) increased the differentiation of rat stromal-vascular cells into adipocytes, and that hydrated collagen inhibited differentiation.     

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.     

Pathogenicity, Morphology, and Differentiation of Acanthamoeba

Acanthamoeba keratitis is sight threatening corneal infection caused by pathogenic Acanthamoeba. Previous studies have shown the genotypic differences between pathogenic and non-pathogenic species/strains of Acanthamoeba. In this study, we examined the morphological differences between pathogenic and non-pathogenic species/strains using scanning electron microscopy. Pathogenic Acanthamoeba exhibited higher number of acanthopodia (structures associated with the binding of amoeba to the target cells) as compared to non-pathogens. In addition, interactions of amoeba with the corneal epithelial cells were studied. Only pathogenic amoeba exhibited adhesion to epithelial cells. Further results indicated that phagocytosis occurs in the pathogenic amoeba by the formation of amoebastome (characteristic of amoeba phagocyte). This study showed that Acanthamoeba phagocytosis may be both an efficient means of obtaining nutrients for the amoeba and a significant factor in the pathogenesis of Acanthamoeba infections. Received: 2 April 2001 / Accepted: 12 April 2001     

Expression of the apoptosis inducer gene head involution defective in primordial germ cells of the Drosophila embryo requires eiger,p53, and loki function

Nanos (Nos) is an evolutionarily conserved protein essential for the maintenance of primordial germ cells (PGCs). In Drosophila, the PGCs or pole cells express head involution defective (hid), which is required for caspase activation, but its translation is repressed by maternal Nos. In the absence of Nos activity, translation of hid mRNA into protein induces apoptosis in pole cells. However, it remains unclear how hid mRNA is regulated in pole cells. Here, we report that hid expression requires eiger (egr), a tumor necrosis factor ligand (TNF) homologue, which is induced in pole cells by decapentaplegic (dpp). In addition, we demonstrate that p53 and loki (lok), a damage‐activated kinase known to be required for p53 phosphorylation, are both required for hid expression in pole cells. Since maternal lok mRNA is enriched in pole cells, it is possible that ubiquitously distributed p53 is activated in pole cells by maternal Lok. We propose that hid expression is activated in a pole cell‐specific manner by loki/p53 and dpp/egr during embryogenesis.     

Orientation of the photosynthetic flagellate,Peridinium gatunense,in hypergravity

The photosynthetic freshwater flagellate,Peridinium gatunense, uses both positive phototaxis and negative gravitaxis to move upwards in the water column. At higher fluence rates approaching those at the surface of their habitat, the cells tend to become unoriented and thus stop their upward movement. Orientation and motility ofPeridinium gatunense has been studied in the slow rotating centrifuge microscope (NIZEMI), which allows observation of swimming behavior during centrifugation acceleration between 1g and 5g. The movement vectors were analyzed by real time image analysis capable of tracking many cells simultaneously. At 1g the orientation was not very precise, but the degree of orientation increased significantly at higher acceleration forces up to about 3g. Most cells were capable of swimming even against an acceleration vector of 3.8g; at higher acceleration forces the cells were not able to cope with the centrifugal force. The linear velocity of cells swimming against 1g was about 20% lower than that of cells moving in other directions. The velocity decreased even more in cells swimming against higher acceleration forces.     

LrrkA,a kinase with leucine‐rich repeats,links folate sensing with Kil2 activity and intracellular killing

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine‐rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.     

Mitogenicity and Adjuvanticity of a Marine Bacterium,Vibrio anguillarum,in Mice

The effects of whole cells of three different O serotypes of Vibrio anguillarum on the murine immune response were studied. The addition of different doses (1–100/ig/ml) of V. anguillarum cells, as well as Salmonella typhimurium lipopolysaccharide, markedly increased the incorporation of [³H] thymidine into in vitro cultured spleen cells of C57BL/6 mice. All three serotype strains of V. anguillarum were able to induce the mitogenic effect at 10 μg/ml and 100 μg/ml, but serotype I strains were more potent than the others. Since pretreatment of spleen cells with rabbit anti-mouse thymocyte antiserum did not affect the mitogenic activity of V. anguillarum, Vibrio cells may be a B-lymphocyte mitogen. When sheep or horse erythrocytes and Vibrio cells were injected intraperitoneally into ddY mice, Vibrio cells exhibited an enhancing effect on antibody response in vivo, regardless of the different serotypes. Vibrio cells, when injected intraperitoneally into mice before the antigen, markedly suppressed the antibody response. Several days after the injection of Vibrio cells, these mice showed an enhanced carbon clearance activity. Acid phosphatase activity in their peritoneal cells was also augmented, suggesting that Vibrio cells activated macrophages in the mice.     

The Response of Prolactin,ACTH, and Growth Hormone Cells in the Pituitary Gland of the Three-Spined Stickleback,: Gasterosteus aculeatus L. form leiurus,to Increased Environmental Salinities

Benjamin, M. 1980. The response of prolactin, ACTH, and growth hormone cells in the pituitary gland of the three-spined stickleback, Gasterosteus aculeatus L. form leiurus, to increased environmental salinities. (Department of Anatomy, University College, Cardiff, Wales, U.K.) — Acta zool. (Stockh.) 61(1): 1–7. The time-sequence of response of the prolactin, ACTH and growth hormone cells in the pituitary gland of the leiurus form of the three-spined stickleback, Gasterosteus aculeatus L., to a transfer from freshwater to seawater, was studied by light microscopy. The appearance of the pituitary was compared with that of animals caught in brackish or sea water. The prolactin cells respond only slowly to seawater by cytological changes interpreted as signs of decreased secretory activity. It is thus suggested that prolactin is unlikely to be important for osmoregulation in this stickleback. The growth hormone cells are more active in all seawater-adapted animals, while the ACTH cells are less active—although only in laboratory experiments. The differing responses of the pituitaries of the leiurus and trachurus forms of G. aculeatus and Pungitius pungitius to high salinities are compared. Even in species whose pituitaries are virtually identical, the response to high salinites may differ widely.     

Fluorescent probe studies of normal, persistently infected, rous sarcoma virus-transformed, and trypsinized rat cells

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine were used to study membranes of normal cells, RSV-transformed cells, cells treated with a proteolytic enzyme, and cells persistently infected with lymphocytic choriomeningitis virus. The lifetimes of excited pyrene and pyrene butyric acid showed only minor changes when these probes were in normal, transformed, trypsinized or persistently infected cells. However, pyrene, but not pyrene butyric acid, lifetimes are shorter in cell membranes than in homogeneous solvents. The quenching of excited pyrene in cells by quencher molecules was slower than corresponding reactions in homogeneous solutions indicating that the probe was screened from the quenchers by the membrane. However, quenching reactions with the pyrene butyric acid probe were similar in cells and homogeneous solvents. This indicates that pyrene and pyrene butyric acid reside in different lipid regions of the membrane. Transformed and trypsinized cells showed increased membrane fluidity compared to normal and persistently infected cells. Membrane fluidity was determined from the excimer/monomer fluorescence ratios of pyrene, and by the polarization of N-phenyl 1-naphthylamine fluorescence. Several techniques distinguished between normal and transformed or trypsinized cells; however, the only parameter unique to viral transformation was a blue shift of the fluorescence maxima of N-phenyl 1-naphthylamine. This shift reflected a less polar environment for N-phenyl 1-naphthylamine in virus-transformed cells.     

LAURENCIA MARILZAE SP. NOV. (CERAMIALES,RHODOPHYTA) FROM THE CANARY ISLANDS,SPAIN, BASED ON MORPHOLOGICAL AND MOLECULAR EVIDENCE1

Laurencia marilzae Gil‐Rodríguez, Sentíes et M.T. Fujii sp. nov. is described based on specimens that have been collected from the Canary Islands. This new species is characterized by distinctive yellow–orange as its natural habitat color, a terete thallus, four pericentral cells per vegetative axial segment, presence of secondary pit‐connections between adjacent cortical cells, markedly projecting cortical cells, and also by the presence of corps en cerise (one per cell) present in all cells of the thallus (cortical, medullary, including pericentral and axial cells, and trichoblasts). It also has a procarp‐bearing segment with five pericentral cells and tetrasporangia that are produced from the third and fourth pericentral cells, which are arranged in a parallel manner in relation to fertile branchlets. The phylogenetic position of this taxon was inferred based on chloroplast‐encoded rbcL gene sequence analyses. Within the Laurencia assemblage, L. marilzae formed a distinctive lineage sister to all other Laurencia species analyzed. Previously, a large number of unique diterpenes dactylomelane derivatives were isolated and identified from this taxon. L. marilzae is morphologically, genetically, and chemically distinct from all other related species of the Laurencia complex described.     

Silver,copper, lead and zinc accumulation byPseudomonas stutzeri AG259 andStreptomyces albus : electron microscopy and energy dispersive X-ray studies

Metal accumulation by a silver-resistant Pseudomonas stutzeri AG259 strain and a Streptomyces albus strain was investigated in a mixed metal solution of silver, copper, lead and zinc. The location of silver, lead and copper on cells was determined by transmission electron microscopy coupled with an X-ray analysis system. In P. stutzeri cells silver was detected as dense deposits on the cells. Copper and lead were distributed over the cells. S. albus accumulated these metals only on part of cells with a higher concentration per cell than in P. stutzeri.     

TETRASPORA,CHLOROSACCUS, AND PHAEOSPHAERA,A UNIQUE EXAMPLE OF PARALLEL EVOLUTION IN THE ALGAE1

The similarity among Tetraspora, Chlorosaccus, and Phaeosphaera, belonging to the Chlorophyceae, Xanlhophyceae, and, Chrysophyceae, adds to the concept of parallel evolution among algal classes. Not only are all 3 genera characterized by colonial organization, nonmotile vegetative cells embedded in a gelatinous matrix, but the genera are also found in the same habitat. Further, the characteristic pseudocilia of Tetraspora cells are also found in colonics of Phaeosphaera. Morphological features of pseudocilia of Tetraspora and the nature of papillae in Chlorosaccus are also presented.     

Ultrastructure of the lycophora larva of Gyrocotyle urna (Cestoda,Gyrocotylidea)

Summary The ultrastructure of the protonephridial system of the lycophore larva of Gyrocotyle urna Grube and Wagener, 1852, is described. It consists of six terminal cells, at least two proximal canal cells, two distal canal cells and two nephridiopore cells. The terminal cells and the proximal canal cell build up the filtration weir with its two circles of weir rods. The proximal canal cell constitutes a solid, hollow cylinder without a cell gap and desmosome. The distal canal cell is characterized by a strong reduction of the canal lumen by irregularly shaped microvilli. The nephridiopore region is formed by a nephridiopore cell; its cell body is located at some distance proximally within the larva. The connection among different canal cells is brought about by septate desmosomes. Morphological, evolutionary and functional aspects of the protonephridial system within Platyhelminthes are discussed. The structure of the proximal canal cells without a desmosome is considered an autapomorphy of Cestoda.Abbreviations ci cilia of the terminal cell - Co distal canal cell - col lumen of the distal canal cell - Ep epidermis - er outer rods of the filtration weir - il inner leptotriches - ir inner rods of the filtration weir - ld lipid droplets - mt microtubule - mv microvilli - Nc nephridiopore cell - Ne neodermis anlage cells - nu nucleus - pC proximal canal cell - ro ciliary rootlets - sd septate desmosome - Tc terminal cell     

Statins,stem cells,and cancer

The statins (3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors) were proven to be effective antilipid agents against cardiovascular disease. Recent reports demonstrate an anticancer effect induced by the statins through inhibition of cell proliferation, induction of apoptosis, or inhibition of angiogenesis. These effects are due to suppression of the mevalonate pathway leading to depletion of various downstream products that play an essential role in cell cycle progression, cell signaling, and membrane integrity. Recent evidence suggests a shared genomic fingerprint between embryonic stem cells, cancer cells, and cancer stem cells. Activation targets of NANOG, OCT4, SOX2, and c‐MYC are more frequently overexpressed in certain tumors. In the absence of bona fide cancer stem cell lines, human embryonic stem cells, which have similar properties to cancer and cancer stem cells, have been an excellent model throwing light on the anticancer affects of various putative anticancer agents. It was shown that key cellular functions in karyotypically abnormal colorectal and ovarian cancer cells and human embryonic stem cells are inhibited by the statins and this is mediated via a suppression of this stemness pathway. The strategy for treatment of cancers may thus be the targeting of a putative cancer stem cell within the tumor with specific agents such as the statins with or without chemotherapy. The statins may thus play a dual prophylactic role as a lipid‐lowering drug for the prevention of heart disease and as an anticancer agent to prevent certain cancers. This review examines the relationship between the statins, stem cells, and certain cancers. J. Cell. Biochem. 106: 975–983, 2009. © 2009 Wiley‐Liss, Inc.     

MOLYBDENUM DEPENDENCE,NITRATE UPTAKE AND PHOTOSYNTHESIS OF FRESHWATER PLANKTON ALGAE1,2

The effects of molybdenum on photosynthesis and nitrate uptake by two species of freshwater algae, Navicula pelliculosa (Bréd.) Hilse and Chlamydomonas reinhardtii Dang. Were examined. Photosynthetic rates in cells of N. Pelliculosa deprived of Mo for 48 h were significantly lower than in nondeprived cells; the rates were 2.6 and 4.5 μg C/10⁶ cells/h, respectively. There was no significant reduction in photosynthetic rates of C. reinhardtii under the 2 treatments, the rates were both ca. 6.0 μg C/10⁶ cells/h. These observed rates in the 2 species were not altered by added Mo concentrations as high as 1.0 ppm. The chlorophyll a values in 48 h Mo-deprived cells were 1.15 μg/10⁶ cells compared to 1.57 μg/10⁶ cells provided with this metal. Molybdenum deficiency did not affect the chlorophyll a concentration in Chlamydomonas; the chlorophyll levels were 2.44μg/10⁶ cells. Nitrate uptake by Mo-deprived cells of N. Pelliculosa was significantly lower than in cells cultured in Mo; the rates were 0.087 and 0.238 μM NO_a⁻/10⁶ cells/h, respectively. Uptake rates by C. reinhardtii were similar with or deprived of Mo. The K_m values for No₃⁻ uptake were 14.9 and 148.0 μM for N. Pelliculosa and C. reinhardtii, respectively.