Chloroquine and Hydroxychloroquine Increase Retinal Pigment Epithelial Layer Permeability

Antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used as antiinflammatory drugs, but side effects include retinopathy and vision loss. The objective of this study was to examine the effect of CQ and HCQ on the barrier integrity of retinal pigment epithelial (RPE) cell monolayers in vitro. Permeability of ARPE‐19 cell monolayers was determined using Fluorescein isothiocyanate (FITC)‐labeled dextran. The influence of CQ and HCQ on cell death and the expression tight junction molecules was examined. CQ and HCQ significantly increased ARPE‐19 monolayer permeability after 3 and 18 h, respectively, and enhanced mRNA levels for claudin‐1 and occludin. Cytotoxicity was only observed after 18 h exposure. Thus, CQ and HCQ rapidly enhance RPE barrier permeability in vitro, independent of cytotoxicity or loss of zonula occludens‐1, claudin‐1, and occludin expression. Our findings suggest that CQ/HCQ‐induced permeability of the RPE layer may contribute to blood–retinal barrier breakdown in case of CQ/HCQ‐induced retinopathy.     

Immunogenicity of Ehrlichia ruminantium Grown in Tick Cell Lines

Ehrlichia (previously Cowdria) ruminantium, the pathogen which causes heartwater in domestic and wild ruminants, can now be propagated in cell lines from one vector (Amblyomma variegatum) and five non-vector (Ixodes scapularis, I. ricinus, Boophilus decoloratus, B. microplus and Rhipicephalus appendiculatus) tick species. E. ruminantium isolates from West and South Africa and the Caribbean vary in their cell line preference, growth patterns and immunogenic capability. In laboratory trials, certain combinations of tick cell line and E. ruminantium isolate were highly immunogenic in sheep. These trial vaccines were grown under specific in vitro conditions and administered as a single intravenous dose of freshly harvested whole, live culture. Following immunisation and subsequent exposure to virulent E. ruminantium, protected sheep showed no clinical response and a range of serological responses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neuronatin对人视网膜色素上皮细胞增殖迁移的影响

目的:研究胚胎时期表达部位广泛、丰度高,而成年后分化表达的印记基因Neuronatin(Nnat)的两种剪接形式Nnatα和Nnatβ对人视网膜色素上皮细胞(RPE)增殖、迁移的影响。方法:构建Nnatα、β两种剪接形式的表达质粒,转染RPE获得表达该基因的稳定表达细胞株;CCK-8实验检测稳定表达细胞株的增殖能力,流式细胞仪分析细胞周期,细胞划痕实验检测其迁移能力。结果:成功构建了Nnatα和Nnatβ表达质粒,并获得了Nnatα和Nnatβ基因稳定表达PRE细胞株。CCK-8实验结果显示cNNATα组与对照组相比较,增值率为23.33%(P0.05),cNNATβ组相较于对照组无显著性差异,细胞周期分析cNNATα组和cNNATβ组细胞在G2-S期的百分率分别为18.60%、11.11%,对照组细胞的为9.94%;相较于对照组,cNNATα组的细胞迁移能力显著增强,cNNATβ组的细胞迁移能力微弱增强。结论:Nnatα对RPE有一定的增殖作用,其影响主要在S期;同时,Nnatα显著促进RPE细胞的迁移能力。     

Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity

The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor susceptibility gene 101 (Tsg101), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of Tsg101 severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.     

Kinetics of Experimental Infection of Sheep with Ehrlichia ruminantium Cultivated in Tick and Mammalian Cell Lines

Inoculation of sheep with Ehrlichia (previously Cowdria) ruminantium which has been cultivated in mammalian endothelial cell cultures is almost always followed by a severe clinical reaction, whereas inoculation of the agent cultivated in tick cell lines usually does not provoke a clinical response, but may result in seroconversion and/or protection against subsequent challenge with virulent stabilates. A quantitative, real-time PCR assay was developed to determine the kinetics of infection (rickettsaemia) in sheep inoculated with tick cell- and mammalian cell-derived E. ruminantium (Gardel isolate). The method and initial results are described, and the significance of the findings is discussed in relation to the clinical responses of the sheep. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

CO2 Dynamics and Growth in Photoautotrophic and Photomixotrophic Apple Cultures

The daily dynamics of CO₂ concentration in the culture vessels and the photoautotrophic or photomixotrophic growth capacity of apple (Malus pumila hybrid MM 106 paradisiaca× Northern Spy) cultures were studied. The photoautotrophic cultures were grown on a sugar-free growth medium and submitted (0S+CO₂) or not (0S-CO₂) to periodic injections of exogenous CO₂. The photomixotrophic cultures were grown in the presence of 30 g dm⁻³ sucrose, with (30S+CO₂) or without (30S-CO₂) CO₂ enrichment. The photosynthetic photon flux density applied was of 210 ± 5 μmol m⁻²s⁻¹. In the 0S-CO₂ treatment, CO₂ showed rather uniform and narrow light-dark fluctuations throughout the culturing cycle. In the 30S-CO₂ treatment, the daily ratio between CO₂ produced during the dark period and that uptaken during the following light period, was almost always above 1 with the only exception of a few days (from the 5^th to the 9^th day) when the amount of photosynthesised CO₂ was equal to or higher than that produced during dark respiration. The 0S+CO₂ cultures needed to be enriched all days with exogenous CO₂ to avoid periods of gas deficiency while in 30S+CO₂ the CO₂ injected the first culturing day was uptaken over 5 d; thereafter, daily injections were necessary. Culture fresh and dry mass, number of newly formed shoots and number of nodes per shoot in 0S+CO₂ treatment did not statistically differ from the values obtained with 30S−CO₂. The highest growth was observed in 30S+CO₂ treatment. The increase in culture fresh mass due to 1 μmol of CO₂ added to the culture vessels was 1.54 and 1.36 mg for 30S and 0S respectively, while in terms of dry mass the increase was about 2.5 times higher in the sugar-enriched treatment. CO₂ enrichment accounted for 77.3 % and 21.2 % of the final fresh mass in 0S+CO₂ and 30S+CO₂, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Permeation of Four Oral Drugs Through Human Intestinal Mucosa

The pharmaceutical industry is in need of rapid and accurate methods to screen new drug leads for intestinal permeability potential in the early stages of drug discovery. Excised human jejunal mucosa was used to investigate the permeability of the small intestine to four oral drugs, using a flow-through diffusion system. The four drugs were selected as representative model compounds of drug classes 1 and 3 according to the biopharmaceutics classification system (BCS). The drugs selected were zidovudine, propranolol HCl, didanosine, and enalapril maleate. Permeability values from our in vitro diffusion model were compared with the BCS permeability classification and in vivo and in vitro gastrointestinal drug permeability. The flux rates of the four drugs were influenced by the length of the experiment. Both class 1 drugs showed a significantly higher mean flux rate between 2 and 6 h across the jejunal mucosa compared to the class 3 drugs. The results are therefore in line with the drugs’ BCS classification. The results of this study show that the permeability values of jejunal mucosa obtained with the flow-through diffusion system are good predictors of the selected BCS class 1 and 3 drugs’ permeation, and it concurred with other in vitro and in vivo studies.     

Tetrapyrrole Accumulation in in vitro Selected Somaclones of Acifluorfen-Tolerant Solanum ptycanthum Dun

Acifluorfen-tolerant callus lines of Solanum ptycanthum were isolated by stepwise selection. Growth of unselected lines was completely inhibited at 0.5 µM acifluorfen, while some selected lines grew at 8 µM acifluorfen. Twenty-two of 25 acifluorfen-tolerant callus lines regenerated shoots. Acifluorfen-tolerant S. ptycanthum callus lines differed in protoporphyrin IX content ranging from 2.0 to 43.5 nmole per 100 mg protein. As the concentration of acifluorfen increased, the amount of protoporphyrin IX accumulated increased. These results indicated that the possible site of action of acifluorfen was protoporphyrinogen oxidase which might be the molecular target of the herbicide within plant cell.     

Factors Affecting Plant Regeneration from Tissue Cultures of Chinese Leymus (Leymus chinensis)

Chinese leymus (Leymus chinensis Trin.) is a perennial grass of the Gramineae, which is widely distributed in China, Mongolia and in Russian-Siberian. In order to explore the potential of biotechnology for genetic improvement of this forage grass, an efficient tissue culture system was established and the factors affecting plant regeneration were evaluated. Immature inflorescence segments 3–5 mm in length from eight accessions were cultured on N6 medium supplemented with 2.26–22.60 µM 2,4-D. The callus induction frequency ranged from 72.11 to 82.19%. Shoots were differentiated from the calli on N6 medium containing 4.65 µM kinetin and 4.44 µM BA. Viable regenerants were developed on hormone-free medium. Normal plants were obtained after natural vernalization in the field. The plant regeneration frequency in Chinese leymus was associated with different genotypes and different combinations of growth regulators in medium. The concentration of 2,4-D in the callus induction medium had a strong effect on successive plant regeneration. Relatively higher concentrations of 2,4-D (i.e., 9.04 and 22.60 µM) were more favorable to the plant regeneration than lower ones (i.e., 2.26 and 4.52 µM). This is the first report on plant regeneration in vitro in L. chinensis.     

Regeneration from Phylloclade Explants and Callus Cultures of Schlumbergera and Rhipsalidopsis

Phylloclade explants of Schlumbergera and Rhipsalidopsis were cultured in vitro to produce axillary and adventitious shoots. The explants of both species, taken from greenhouse-grown plants, produced only axillary shoots. There was a pronounced improvement in adventitious shoot formation in phylloclade explants of cultivar CB4 of Rhipsalidopsis by increasing numbers of subcultures of axillary shoots used as donor plants. The axillary shoots generated from the explants were either subcultured to produce successive generations of axillary shoot cultures or made into phylloclade explants and tested for adventitious shoot formation at each subculture. The duration of each subculture varied from 6 to 12 weeks. After the first subculture, sporadic adventitious shoot formation began, and after the third subculture 87% explants of cultivar CB4 produced adventitious shoots at a frequency of about 12 shoots per explant. In contrast, there was no improvement in regenerative ability in explants of cultivar Thor-Olga of Schlumbergera up to third subculture. Adventitious shoots could be produced by callus culture too. Cultivar CB4 was highly regenerative, producing as many as 10 adventitious shoots per square cm of callus. In vitro grown plantlets, when transferred to pots continued to show prolific growth.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.     

Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu²⁺-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies.     

Maintenance of Functional Stem Cells in Isolated and Cultured Adult Intestinal Epithelium

We have previously described a method for the primary culture of adult large intestinal epithelium, suggesting that stem cells had survived both the isolation and the culture procedures. However, as no markers for such cells exist, confirmation of stem cell survival is difficult-only the functional properties can be used to define them. Unfortunately, many of these (e.g., differentiation, crypt regeneration) do not occur in culture, probably due to suboptimal conditions. To address this problem both freshly isolated and cultured small and large intestinal crypts were grown subcutaneously in an immunocompromized mouse. All initially formed cysts lined by a simple epithelium which gradually became multicellular and formed invaginations containing many mitoses and apoptoses. Epithelial differentiation, as assayed by Goblet cell mucin production, was also apparent. Mucin maturation was also typical of the normal intestine. The lumen was frequently filled with mucin and apoptotic bodies. Interestingly, in grafts displaying pronounced crypt-like morphology the regions of proliferation were situated toward the base of the structure and the Goblet cells toward the lumen, i.e., a typical crypt-like morphology. Hence, functional adult stem cells appear to survive isolation and tissue culture, permitting organotypic regeneration, possibly involving homeobox gene expression. This may now allow direct stem cell characterization and experimental manipulation, such as transfection, and may ultimately permit transplantation and therapeutic gene therapy.     

Growth and Enzyme Activity in Roots and Calli of Resistant and Susceptible Allium Lines as Influenced by Sterile Culture Filtrates of Phoma terrestris

Growth and activities of peroxidases, chitinases and glucanases were studied in order to evaluate the response of calli and roots of pink root-susceptible Allium cepa cvs. Valcatorce and T-412 and resistant A. fistulosum cv. Nogiwa Negi, to sterile culture filtrates of Phoma terrestris. Untreated calli and roots of A. fistulosum exhibited higher activity of peroxidases and glucanases than that of Valcatorce and T-412. Enzyme activities and growth of roots and calli were not affected in filtrate-treated A. fistulosum. The growth of calli and roots of A. cepa cultivars decreased significantly after exposure to P. terrestris filtrates while the peroxidase and glucanase activities increased. Peroxidase and glucanase activities were also enhanced in roots of Valcatorce bulbs grown in P. terrestris-inoculated soil as compared to healthy control plants. We conclude that a high constitutive activity of glucanases and perhaps chitinases might account for the resistance of A. fistulosum. The differential reaction (with respect to root growth) of pink root-susceptible and resistant materials to culture filtrates indicates that this in vitro-system might be useful for the screening of onion breeding lines.     

Toxicity testing of polymer materials for dialysis equipment: is there any need forin vivo testing?

In an earlier work, slightly more than 650 plastic materials, intended for use in medical devices, were tested with a battery of chemical, as well asin vitro andin vivo biological tests. An analysis showed that only a limited number of the tests used were actually necessary to obtain the same pass or fail decision as that obtained using the full test battery. This prompted us to prescreen all new materials with a small test battery consisting of the two most discriminating chemical tests and anin vitro cell growth inhibition test. The present work is a report of our findings after testing another 155 materials using this prescreen system.For each single one of the 155 tested materials the same decision on whether or not to use the material in the intended medical device would have been reached without anyin vivo testing. In no single case in a total of 851in vivo tests did an eluate that had passed thein vitro cell test give rise to a reactionin vivo. Thus, among the tests on living systems the cell test alone seems to be sensitive enough to provide sufficient information. Nothing appears to be gained from thein vivo animal tests. However, some of the materials that passed the prescreening tests later failed in one or several of the chemical tests. Both nonspecific chemical tests and tests for specific molecules seem to detect undesirable levels of leachable substances not detected by the prescreening system. Therefore these tests should not be abandoned. Abandoning unnecessaryin vivo testing, on the other hand, would save considerable costs.     

Micropropagation of Bauhinia variegata and Parkinsonia aculeata from nodal explants of mature trees

In vitro propagation protocols were established for two leguminous trees, Bauhinia variegata and Parkinsonia aculeata. In each case axillary shoot proliferation was achieved from nodal explants from mature (6-2-8 years) trees using Murashige & Skoog's medium supplemented with 2.22–31.1 M of 6-benzyladenine. Subsequent rooting of the regenerated shoots was achieved on medium containing 2.46–14.8 M of indole-3-butyric acid. Successful transfer of the regenerants to soil has been accomplished.     

Somatic embryogenesis in plantain banana

Summary A cell suspension of French Sombre plantain banana (Musa spp. AAB genome) was initiated from callus obtained from young male flowers. Histological examination enabled us to describe and follow the evolution of the suspension consisting of: embryogenic aggregates, proembryos, nodules, and isolated cells. It demonstrated the unicellular origin of somatic embryos, either during maintenance of the suspension or after plating on a semisolid medium. The cells from which the embryos originated had no starch but only protein reserves. Plating 1 ml of packed cells from the suspension led to the formation of 10⁵ embryos of which 10 to 40% could be converted into plantlets.