Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro

Summary A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65–75%) and goblet cells (25–35%). Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5±0.5×10⁴ cells) were incubated with toxin dilutions (0.01–20 μg) for 1 h at 24° C at a final pH of 7.8. The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5–5 μg). Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5–10 μg. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar. Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.     

Anin vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells

Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. Thein vitro cytotoxicity of chlorhexidine was evaluated with the Smulow-Glickman (S-G) gingival epithelial cell line. The potency of chlorhexidine was dependent on the length of exposure and composition of the exposure medium. The midpoint cytotoxicity values for 1-, 24-, and 72-h exposures were 0.106, 0.011, and 0.0045 mmol/L, respectively. S-G cells exposed for 2 h to chlorhexidine and then maintained for 48 h in chlorhexidine-free medium were unable to recover from the initial insult. The adverse effects of chlorhexidine on the plasma membrane were suggested by the leakage of lactic acid dehydrogenase from chlorhexidine-treated S-G cells and by the increased permeability of chlorhexidine-treated liposomes to Ca²⁺. The toxicity of a 24-h exposure to chlorhexidine to the S-G cells was progressively lessened as the content of fetal bovine serum (FBS) in the exposure medium was increased from 2% to 8%. The potency of a 1-h exposure to chlorhexidine was reduced in medium amended with albumin, lecithin, and heat-killedEscherichia coli. These reductions in toxicity were presumably due to the binding of the cat onic chlorhexidine to the negatively charged chemical moieties of the components of FBS and of albumin and lecithin and of sites on the surfaces of bacteria. Combinations of chlorhexidine and carbamide peroxide were additive in their cytotoxicities.Abbreviations ANOVA analysis of variance - [Ca²⁺]i calcium concentration in internal medium of liposomes - DMEM Dulbecco's modified Eagle medium - EDTA ethylenediamine tetraacetic acid - FBS fetal bovine serum - Hepes N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid - HGF-1 human gingival fibroblast cell line - HSD honestly significant differences - KB cell line derived from a human epidermoid carcinoma in the mouth - LDH lactic acid dehydrogenase - NADH nicotinamide adenine dinucleotide, reduced form - NR neutral red - NR₅₀ concentration inhibiting neutral red uptake by 50% - PBS phosphate-buffered saline - SEM standard error of the mean - S-G Smulow-Glickman human gingival epithelial cell line     

Rapid chemosensitivity assay with human normal and tumor cells in vitro

Summary Neutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development. This work has been supported, in part, by funds from Schering Corporation, New Jersey, and Chevron Environmental Health Center, Inc., California.     

Rhodamine 123 as a probe of in vitro toxicity in MDCK cells

The effect of mercuric chloride on Madin-Darby Canine Kidney (MDCK) cells grown in culture was assayed by the mitochondrial-specific fluorescent probe, rhodamine 123. Treatment of cells with mercuric chloride resulted in a dissipation of rhodamine fluorescence from the mitochondria into the cytoplasm, followed by a release into the medium bathing the cells. Toxicity was assayed either by determining the proportion of cells with delocalized rhodamine fluorescence, or by measuring the rhodamine released from or retained in the cells. Quantifying the release or retention of rhodamine 123 is semi-automated and represents a highly sensitive method of using a vital fluorescent dye for in vitro toxicity analysis.     

Tentative determination of the molecular weight of substances stimulating the flowering of winter wheat

Murashige & Skoog nutrient was supplemented with substances of molecular weight (M_W) less than 5 kDa, which were separated from extract of winter wheat ears by means of Sephadex G-25 ultrafiltration. Isolated embryos of the same wheat cultivar (Grana) were vernalized in the nutrient for 0 and 7 days at 2 °C for 2 weeks and planted in a glass-house. After 150 days of growth (20/17 °C day/night) the development of the shoot apices was observed. It was found that substances of M_W<5 kDa strongly stimulated the generative development of the plants, enabling the earing of 30 % of non-vernalized plants (control=0%) and 100 % plants vernalized for 7 days (control=29 %). The substances present in the extract of both spring (cv. Jara) and winter (cv. Grana) varieties were fractionated by means of Sephadex chromatography into 300 fractions of M_W=1 to 5 kDa and each of them was added to the isolated embryos of cv. Grana. The embryos were vernalized at 2 °C for 7 days and then cultured as previously described. It was found that the differentiation of the shoot apices was stimulated by over 34 % by fractions of winter wheat extract and more than 50 % by fractions of the spring wheat extract. However, only a few of identical fractions of the extracts of both wheat varieties were able to induce the earing of the plants. These fractions were grouped in 4 continuous intervals of M_W equal to about 4.5–4.9, 3.2–3.3, 2.1–2.6 and 1.00–1.03 kDa. Within the three intervals was identified a small group of identical fractions, which affected the growth of the seedlings in similar mode i.e. inhibiting or stimulating. Thus it can be assumed that these intervals contained identical or similar substances capable of stimulating strongly the earing of winter wheat.     

Leupaxin binds to PEST domain tyrosine phosphatase PEP

PEST domain tyrosine phosphatase (PEP) is an intracellular protein tyrosine phosphatase and characterized by PEST motifs and proline-rich domains in the carboxyl terminal half. PEP is primarily expressed in hematopoietic cells, and together with PEP-binding Csk, may act as a negative regulator of antigen receptor signaling in lymphocytes. Here, we show the binding capability of PEP for leupaxin, which is preferentially expressed in hematopoietic cells and a comparatively new member of the paxillin family characterized by two protein-protein interaction modules, LIM domains and LD motifs. These results suggested that leupaxin might participate in the regulation of the signaling cascade through the binding to PEP in lymphocytes. (Mol Cell Biochem 269: 13–17, 2005)     

Extracellular secretion of phenolic substances from living brown algae

The chemical structures of ultraviolet (UV)- absorbing substances secreted from the healthy living brown algae, Eisenia bicyclis and Ecklonia kurome, were demonstrated. The living activity of algal cells was critically examined using a confocal laser-scanning microscope after incorporation of fluorescein diacetate (FDA) into the cells. Using thin-layer chromatography (TLC), reversed-phase three-dimensional high-performance liquid chromatography (RP-3D-HPLC) and gas chromatography-mass spectrometry (GC-MS), it was found that the UV-absorbing substances (λ_max 265–270 nm) secreted from the living brown algae mainly consisted of the three monomeric bromophenols, 2,4-dibromophenol, 2,4,6-tribromophenol and dibromo-iodophenol, but not phloroglucinol or phlorotannins. The other minor compounds detected in the secretions were as follows: benzothiazole, fatty acids (14:1, 16:0 and 18:0 acids), franesol, 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate and squalene. Exudation of phloroglucinol and phlorotannins was ascertained to take place only after the cell death of these algae. These results indicate that, whilst the algae are alive, polymeric phlorotannins are strictly kept within the algal body, and only monomeric bromophenols are secreted into the seawater medium.     

Quantitative analysis of antiviral drug toxicity in proliferating cells

The toxicity of most antiviral compounds was dependent on the type of cell used to assay toxicity. Ranking of compounds according to toxicity was, however, very similar (p < 0.01) in the three different cell types used in this study. The difference in toxicity observed for 9--D-arabinofuranosyladenine between Flow 5000 cells and CCRF-SB cells could not be accounted for by differences in the intracellular concentrations. On the other hand, the different toxicities observed for ribavirin and 2-deoxy-5-triuorothymidine between Flow 5000 cells and CCRF-SB cells may be caused by the culture conditions (as shown for one cell type, HeLa S₃, grown either as monolayer or in suspension) rather than by cell-specific differences. The growth-inhibitory effect of most antiviral compounds increased with treatment time, indicating an additive nature of toxicity. The ability of cells to recover from toxic treatment with drugs varied greatly from compound to compound (from undetectable regrowth to 140% growth compared to control cells). Coaddition of natural nucleosides could, at best, only partly protect cells from the toxic influences of antiviral nucleoside analogs. As a result of comparing antiviral effects and toxicity in vitro, the unselective compounds may be eliminated from further development at the screening level.Abbreviations ACV 9-(2-hydroxyethoxyethyl)guanine - Ara-A 9--D-arabinofuranosyladenine - Ara-T 9--D-arabinofuranosylthymine - BVDU (E)-5-(2-bromovinyl)-2-deoxyuridine - CCRF-SB human B lymphoblastoid cell line - dA 2-deoxyadenosine - DABG 9-(3,4-dihydroxybutyl)guanine - Flow 5000 human embryo fibroblast cell line - 3FT 3-deoxy-3-fluorodeoxythymidine - dG 2-deoxyguanosine - IDU 5-iodo-2-deoxyuridine - 3T3 Swiss mouse embryo fibroblast cell line - dT 2-deoxythymidine - TFT 2-deoxy-5-trifluorothymidine - PBS phosphate buffered saline solution - PFA trisodium phosphonoformate, foscamet     

In vitro cytotoxicity of heavy metals,acrylamide, and organotin salts to neural cells and fibroblasts

The cytotoxicity of neurotoxic agents was determined for a series of brain-derived cell types and compared with their toxic effects on BALB/c 3T3 fibroblasts, using the neutral red assay. Ranking of toxicants according to their potencies was the same for all cells tested and was in the order of methylmercury>cadmium> mercury>zinc>acrylamide. For a series of di- and triorganotins the ranking order was dibutyl>diphenyl>dibenzyl>diproyl> diethyl>dimethyltin and triphenyl>tribenzyl>trimethyltin, respectively. The test was sensitive enough to detect structure activity relationships between the degree of toxicity and the hydrophobic characteristics of the agents tested.Abbreviations Be₂Sn dibenzyltin dichloride - Be₃Sn triphenyltin dichloride - Bu₂Sn dibutyltin dichloride - CdCl₂ cadmium chloride - CH₃HgCl methylmercuric chloride - DMEM Dulbecco's Modified Eagle's Medium - Et₂Sn diethyltin dichloride - FBS fetal bovine serum - HgCl₂ mercuric chloride - Me₂Sn dimethyltin dichloride - Me₃Sn trimethyltin hydroxide - Phe₂Sn diphenyltin dichloride - Phe₃Sn triphenyltin hydroxide - Pr₂Sn dipropyltin dichloride - ZnCl₂ zinc chloride     

Uptake and transport of organic and inorganic nitrogen by arbuscular mycorrhizal fungi

New information on N uptake and transport of inorganic and organic N in arbuscular mycorrhizal fungi is reviewed here. Hyphae of the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) were shown to transport N supplied as ¹⁵N-Gly to wheat plants after a 48 h labelling period in semi-hydroponic (Perlite), non-sterile, compartmentalised pot cultures. Of the ¹⁵N supplied to hyphae in pot cultures over 48 h, 0.2 and 6% was transported to plants supplied with insufficient N or sufficient N, respectively. The increased ¹⁵N uptake at the higher N supply was related to the higher hyphal length density at the higher N supply. These findings were supported by results from in vitro and monoxenic studies. Excised hyphae from four Glomus isolates (BEG 84, 107, 108 and 110) acquired N from both inorganic (¹⁵NH₄ ¹⁵NO₃, ¹⁵NO₃ ⁻ or ¹⁵NH₄ ⁺) and organic (¹⁵N-Gly and ¹⁵N-Glu, except in BEG 84 where amino acid uptake was not tested) sources in vitro during short-term experiments. Confirming these studies under sterile conditions where no bacterial mineralisation of organic N occurred, monoxenic cultures of Glomus intraradices Schenk and Smith were shown to transport N from organic sources (¹⁵N-Gly and ¹⁵N-Glu) to Ri T-DNA transformed, AM-colonised carrot roots in a long-term experiment. The higher N uptake (also from organic N) by isolates from nutrient poor sites (BEG 108 and 110) compared to that from a conventional agricultural field implied that ecotypic differences occur. Although the arbuscular mycorrhizal isolates used contributed to the acquisition of N from both inorganic and organic sources by the host plants/roots used, this was not enough to increase the N nutritional status of the mycorrhizal compared to non-mycorrhizal hosts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ectomycorrhizal responses to organic and inorganic nitrogen sources when associating with two host species

While it is established that increasing atmospheric inorganic nitrogen (N) deposition reduces ectomycorrhizal fungal biomass and shifts the relative abundances of fungal species, little is known about effects of organic N deposition. The effects of organic and inorganic N deposition on ectomycorrhizal fungi may differ because responses to inorganic N deposition may reflect C-limitation. To compare the effects of organic and inorganic N additions on ectomycorrhizal fungi, and to assess whether host species may influence the response of ectomycorrhizal fungi to N additions, we conducted an N addition experiment at a field site in the New Jersey pine barrens. Seedlings of two host species, Quercus velutina (black oak) and Pinus rigida (pitch pine), were planted at the base of randomly-selected mature pitch pine trees. Nitrogen was added as glutamic acid, ammonium, or nitrate at a rate equivalent to 227.5 kg ha⁻¹ y⁻¹ for eight weeks, to achieve a total application of 35 kg ha⁻¹ during the 10-week study period. Organic and inorganic N additions differed in their effects on total ectomycorrhizal root tip abundance across hosts, and these effects differed for individual morphotypes between oak and pine seedlings. Mycorrhizal root tip abundance across hosts was 90 % higher on seedlings receiving organic N compared to seedlings in the control treatment, while abundances were similar among seedlings receiving the inorganic N treatments and seedlings in the control. On oak, 33–83 % of the most-common morphotypes exhibited increased root tip abundances in response to the three forms of N, relative to the control. On pine, 33–66 % of the most-common morphotypes exhibited decreased root tip abundance in response to inorganic N, while responses to organic N were mixed. Plant chemistry and regression analyses suggested that, on oak seedlings, mycorrhizal colonization increased in response to N limitation. In contrast, pine root and shoot N and C contents did not vary in response to any form of N added, and mycorrhizal root tip abundance was not associated with seedling N or C status, indicating that pine received sufficient N. These results suggest that in situ organic and inorganic N additions differentially affect ectomycorrhizal root tip abundance and that ectomycorrhizal fungal responses to N addition may be mediated by host tree species.     

Female Genital Coccidioidomycosis (FGC), Addison's Disease and Sigmoid Loop Abscess Due to Coccidioides Immites; Case Report and Review of Literature on FGC

Nine fungal strains of Dichotomomyces cejpii were studied for their ability to produce antibiotic metabolites. It was found that they produced secondary exo- and endo metabolites with antimicrobial activity against Gram-negative and Gram-positive bacteria, yeasts and moulds and with a toxic effect against animal organs in vitro. Detailed chemical characterization of these active principles need to be carried out. This revised version was published online in June 2006 with corrections to the Cover Date.     

Response of Douglas-fir and amabilis fir to split-root nutrition with inorganic and organic nitrogen

There is limited understanding of the spatial plasticity of conifer root growth in response to inorganic and organic nitrogen (N). In this study, slow-growing amabilis fir and fast-growing Douglas-fir, and slow- and fast-growing seedlots of the latter species were examined for their ability to proliferate roots preferentially in compartments of sand/peat medium enriched in organic and inorganic forms of N. In one experiment, N was supplied as 7.1 or 0.71 mM ammonium, nitrate and ammonium nitrate, and in a second experiment, N was supplied as ammonium or glycine. The seedlings’ ability to compensate for the starvation of a portion of the root system was assessed by measuring biomass of leaves, stems and roots, and foliar N concentration. Both fast- and slow-growing seedlots of Douglas-fir and slow-growing amabilis fir were able to proliferate roots in compartments of soil enriched with inorganic and organic N. In the first experiment, whole plant and root biomass was greatest when N was provided as ammonium followed by nitrate, and in the second experiment, seedling whole and root biomasses did not differ between ammonium and glycine treatments. All seedlings were able to compensate for the starvation of a portion of the root system, thus total plant biomass did not differ between split-root treatments; however, foliar N contents were lower in the 7.1/0.71 mM inorganic N split-root treatments. Foliar N concentrations were also lower in seedlings supplied with glycine.     

Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs)

Unrooted strawberry cv. `Akihime' shoots with three leaves obtained from standard mixotrophic cultures were cultured in the ``Culture Pack'-rockwool system with sugar-free MS medium under CO<sub>2</sub>-enriched condition. To examine the effect of superbright red and blue light-emitting diodes (LEDs) on in vitro growth of plantlets, these cultures were placed in an incubator, ``LED PACK', with either red LEDs, red LEDs1blue LEDs or blue LEDs light source. To clarify the optimum blue and red LED ratio, cultures were placed in ``LED PACK 3' under LED light source with either 100, 90, 80, or 70% red + 0, 10, 20, 30% blue, respectively, and also under standard heterotrophic conditions. To determine the effects of irradiation level, cultures were grown under 90% red LEDs + 10% blue LEDs at 45, 60 or 75 mol m^–2 s^–1 . Plantlet growth was best at 70% red + 30% blue LEDs. The optimal light intensity was 60 mol m^–2 s^–1. Growth after transfer to soil was also best after in vitro culture with plantlets produced were 70% red LEDs + 30% blue LEDs.     

Solubilization of organic and inorganic phosphates by three highly efficient soil bacterial isolates

Screening soil samples collected from a diverse range of slightly alkaline soil types, we have isolated 22 competent phosphate solubilizing bacteria (PSB). Three isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 hydrolyzed inorganic and organic phosphate compounds effectively. Bacterial growth rates and phosphate solubilization activities were measured quantitatively under various environmental conditions. In general, a close association was evident between phosphate solubilizing ability and growth rate which is an indicator of active metabolism. All three PSB were able to withstand temperature as high as 42°C, high concentration of NaCl upto 5% and a wide range of initial pH from 5 to 11 while hydrolyzing phosphate compounds actively. Such criteria make these isolates superior candidates for biofertilizers that are capable of utilizing both organic and mineral phosphate substrates to release absorbable phosphate ion for plants.     

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 2: Rhizogenesis and acclimatisation to ex vitro conditions

Conditions affecting rhizogenesis in vitro and ex vitro and subsequent acclimatisation of Telopea speciosissima (waratah) were investigated. Clonal selections were successfully rooted in vitro in agar, on filter paper bridges or using crushed quartz-sand, the last substrate resulting in superior growth of roots. The in vitro substrates were impregnated with half-strength MS, 7.5 gl^-1 sucrose and various concentrations of IBA. For the quartz-sand, an IBA concentration of 50 M was optimal, 70% of microcuttings were rooted. No plantlets rooted in vitro were acclimatised to ex vitro conditions (using mist, fog or humidity tent regimes). Microcuttings (25–45 mm in length) were rooted ex vitro in a fog humidity regime (droplet size <10 m) using an IBA powder dip (3 g IBA kg^-1). Neither a mist nor a humidity-tent regime was suitable for rooting of waratah microshoots ex vitro. A peat and perlite mixture was superior to crushed quartz-sand or potting mix for the rooting of microshoots; this appeared to be related to the air-filled porosity (>20%) of the mixture, measured after the medium was saturated and then drained for 24h. Plantlets must be left under the high humidity regime until shoot growth resumes (four to eight weeks) otherwise plant mortality increase significantly. In vitro-produced leaves abscised between eight and 12 weeks after transfer to ex vitro conditions, indicating that these structures did not acclimatise ex vitro.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige and Skoog medium     

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.     

Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: A quantitative indicator of metal toxicity

An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of31 metals, 17 were competentfor inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD₅₀ in mice. Apparently the positivelnegative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.Abbreviations hGH human growth hormone - hsp70 70 kDa heat-shock protein