Third Day Of Exposure Research Articles

Water Soluble <i>Pleurotus ostreatus</i> Polysaccharide Down-Regulates the Expression of MMP-2 and MMP-9 in Caco-2 Cells

Many polysaccharides and polysaccharide-protein complexes isolated from mushrooms have immunomodulatory and anti-cancer effects. Our aim was to study the regulatory mechanisms of Caco-2 cell response to water soluble P. ostreatus polysaccharide extract up to 72 hours. Specific enzymatic activities were assessed by kinetic measurements. The reduced glutathione content and the lipid peroxidation level were also analyzed. Protein expression of several heat shock proteins, Bcl-2 and metalloproteinases 2 and 9 were revealed by Western blot. Gelatin zymography assay was used to evaluate the MMP-2 and MMP-9 activities. Until the third day of exposure the total SOD activity decreased continuously by 30%, whereas GST and GR ones diminished by 17% respectively 30.5% compared to control. No significant changes were observed in CAT and G6PDH specific activities as well as in GSH and MDA concentration. After the third day of exposure a significant up-regulation of Hsp60 and Hsp90 expression and a down-regulation of Hsp70 one were registered. Bcl-2 protein levels were down-regulated by 50% in the first day of treatment but increased after 3 days. MMP-2 and 9 secretion in the culture medium was significantly reduced suggesting a diminished ability of invasion of colon cancer cells. Our data revealed that in vitro treatment with P. ostreatus aqueous polysaccharide extract does not induce apoptosis in Caco-2 cell line but it could inhibit the invasion of colon cancer cells through the basement membrane.

Effects of Emissions from an Aluminium Smelter in a Tree Tropical Species Sensitive to Fluoride

Fluoride is among the most phytotoxic atmo- spheric pollutants, commonly linked to the appearance of lesions in susceptible plants around emitting sources. In order to assess the effects of fluoride on leaves of Spondias dulcisParkinson (Anacardiaceae), plants were examined 78 km (non-polluted area) and 0.78 km (pol- luted area) from an aluminium smelter. The level of fluoride increased with the exposure time of the plants in the polluted area. On the third day of exposure in the polluted area, necroses with typical colouration were observed. Micromorphological damage began at the abaxial epidermis, mainly associated with the stomata. Starch grain accumulation was more pronounced in the midrib. The cell membranes and chloroplasts were greatly affected by the pollutant. We observed accumu- lation of phenolic compounds and electron-dense mate- rial at the boundaries of the ending veinlets. The micro- scopic events described precede the appearance of symptoms and are therefore of prognostic value in predicting injury by fluoride and will be useful as biomarkers. The high sensitivity of S. dulcis to fluoride and the specificity of the symptoms confirm, for the first time, in an experiment of active biomonitoring, the potential of this species as a bioindicator.

Temporal analysis of oxidative effects on the pulmonary inflammatory response in mice exposed to cigarette smoke

The most common factor related to the chronic obstructive pulmonary disease (COPD) development is the chronic smoking habit. Our study describes the temporal kinesis of pulmonary cellular influx through BALF analyses of mice acutely exposed to cigarette smoke (CS), the oxidative damage and antioxidative enzyme activities. Thirty-six mice (C57BL/6, 8weeks old, male) were divided in 6 groups: the control group (CG), exposed to ambient air, and the other 30 mice were exposed to CS. Mice exposed to CS presented, especially after the third day of exposure, different cellular subpopulations in BALF. The oxidative damage was significantly higher in CS exposed groups compared to CG. Our data showed that the evaluated inflammatory cells, observed after three days of CS exposure, indicate that this time point could be relevant to studies focusing on these cellular subpopulation activities and confirm the oxidative stress even in a short term CS exposure.

Alteration in Glutathione Homeostasis and Oxidative Stress During the Sequelae of Trimethyltin Syndrome in Rat Brain

Trimethyltin (TMT), a by-product of tin, is used in a wide variety of industrial and agricultural purposes which serves as a model neurotoxicant in hippocampal neurodegeneration, and this could, in turn, be exploited for various therapeutic compounds essential for hippocampal neurodegeneration. Therefore, the present investigation explores the sequential changes in behavior, oxidative burden, and apoptosis following TMT administration in rat hippocampus. Male SD rats weighing 250g were given single dose of 8.5mg/kg TMT (i.p.) that resulted in "TMT syndrome" which begins at the third post-TMT exposure and continued till 21 days posttreatment. This resulted in behavioral alteration (aggression and spontaneous seizures), cognitive impairment as assessed by plus maze, and passive avoidance resulting in short-term memory deficits. These behavioral alterations were associated with an increase in oxidative stress. The levels of malondialdehyde, reactive oxygen species, and protein carbonyl were significantly increased (p < 0.001) in the TMT-treated rats after the third day of exposure and were maximum at day 14 postexposure. The glutathione system was not able to adapt rapidly in response to oxidative stress which resulted in imbalance in redox status. The imbalance in the redox state resulted in the death of neurons as seen by a significant increase in caspase activation at gene as well as protein level after TMT exposure on day 14, quoting an extent of changes. Therefore, it is proposed that behavioral deficits could be accounted by the impairment of endogenous glutathione homeostasis which resulted in death of neurons in the hippocampal region.

Biodegradation of oxytetracycline by Pleurotus ostreatus mycelium: a mycoremediation technique

Oxytetracycline (OTC) is administered in high doses to livestocks and enters the environmental compartments as a consequence of animal waste disposal. As a first step in setting up a useful mycoremediation technique, an OTC lab degradation test was performed in liquid medium using the ligninolytic fungus Pleurotus ostreatus. OTC disappearance in culture medium was clearly evident as early as the third day of exposure onwards, with an almost complete removal after 14d. The drug removal was mediated by fungal absorption in the mycelia, where the OTC molecule underwent a degradation step, as demonstrated by mass spectrometry analyses. A putative degradation product, ADOTC (2-acetyl-2-decarboxamido-oxytetracycline) is proposed. Experimental conditions excluded OTC abiotic degradation; the degradation by extracellular laccase was also experimentally discarded.

The metabolic activity of cells of Fucus vesiculosus Linnaeus, 1753 (Phaeophyta: Fucales) from the Barents Sea under conditions of oil pollution

The metabolic activity of cells of brown seaweed was studied. The metabolic activity of cells of F. vesiculosus increased in an oil-polluted environment. The period of formation of the response to the addition of a toxicant depends on conditions in the algal habitat. Metabolic activity increased during several minutes in samples that were taken from a clean area and on the third day of exposure in samples from an oilpolluted area. The level of metabolic activity of cells of F. vesiculosus can be used as an indicator of the presence of stress factors in the algal habitat.

Medium-term effects of repeated exposure to stray voltage on activity, stress physiology, and milk production and composition in dairy cows

The medium-term effects of permanent or random exposure to stray voltage applied to the water trough were evaluated on milk production and stress physiology in lactating dairy cows. Seventy-four Holstein cows were assigned during two 8-wk experimental periods to 1 of 3 treatments. The treatments were permanent exposure to voltage (PERM, 1.8V, n=23) applied to the water trough, random exposure to voltage (RAND, 1.8V, 36 h/wk, n=25), and no exposure to voltage (control, n=26). On the first day of voltage exposure, PERM cows had higher activity levels than control cows (9.8±2.70 vs. −2.3±2.74 14-s periods of movement/h). During the eighth week of exposure, RAND cows had higher activity levels than control cows (4.2±3.64 vs. −7.7±3.54 14-s periods of movement/h) and higher milk cortisol concentration than PERM cows (0.21±0.024 vs. 0.14±0.020 ng/mL). No differences were observed between treatments for cortisol response after an ACTH challenge during the seventh week of exposure. No effects of voltage exposure were observed on production traits and daily water intake. There was a transient decrease in milk yield on the second day of exposure in PERM cows (−1.4±0.74kg) and on the third day of exposure in RAND cows (−3.5±1.03kg) compared with control cows. In dairy cows, permanent or random exposure to stray voltage (1.8V; 3.6mA) could induce a transient stress response. Moreover, unpredictable voltage exposure could be considered a mild stressor, with slight modifications in stress physiology and activity but no impairment in production in the medium term.

Photosynthetic Response of Maize Plants Against Cadmium and Paraquat Impact

The effects of cadmium (Cd) and/or paraquat (PQ) toxicity on photosynthesis in maize leaves were examined by measurement of gas exchange and chlorophyll content in hydroponically cultured plants. It was found that growth rate was distinctly influenced only by 100 µM Cd treatment. Chlorophyll a and chlorophyll b decreased along with the increase of Cd concentration, while PQ spraying, alone and combined with Cd, increased chlorophyll a content on the third and seventh experimental days. Generally, carotenoid content increased in response to Cd and PQ and reached the highest levels at 100 µM Cd. Rate of photosynthesis in maize decreased after Cd treatment. CO2 assimilation was approximately 60% reduced at 50 µM Cd and 70% reduced in the presence of 100 µM Cd. PQ toxicity was partly overcome after the third day of exposure. Transpiration and stomatal conductance in maize leaves decreased on the third day along with Cd concentration and PQ spraying, except for the 25-µM Cd-treated plants. On the tenth day, the 25-µM Cd-treated plants and those from PQ-treated variants showed an increase of transpiration and stomatal conductance. Maize exhibited an ability to accumulate Cd in high quantities, especially in the roots—over 4,500 mg Cd/kg dry weight.

Toxicity and persistence of sodium fluoroacetate (1080) in the land crab (Gecarcinus lagostoma) on Ascension Island

An eradication program for introduced feral cats, using sodium fluoroacetate (1080) bait, was planned on Ascension Island to help breeding seabird populations to recover. We investigated the likelihood of mortality and the occurrence of residual 1080 in the ‘non-target’ Ascension land crab (Gecarcinus lagostoma) through simulating ‘realistic’ and ‘worst case’ exposure to 1080 bait. Crabs feeding on 1080 baits ingested an estimated maximum of 9–56 mg 1080 (kg bodyweight)–1 and although two of 32 treatment crabs died, this mortality was not attributed to 1080 poisoning but to other, unknown, causes. Our results suggest that G. lagostoma has relatively low susceptibility to acute toxic effects of 1080. Most residual 1080 was eliminated rapidly from crab tissue, with concentrations of 0.006–0.070 mg (kg bodyweight)–1 measured in crab claw/leg tissue 9–11 days after exposure. Concentrations of 0.200 and 0.650 mg (kg bodyweight)–1 were measured in the claw tissue of two crabs that died from other causes on the third day of exposure to 1080, indicating potential for secondary exposure of sensitive scavengers or predators of 1080-exposed crabs. We recommend a moratorium on human consumption of all crabmeat for a withholding period following the eradication program. The withdrawal period should be defined by further research on the longevity of 1080 in crab tissues, and be confirmed by monitoring of residues in crabs after baiting.

EFEITO DE FUNGOS ANTAGONISTAS E PRODUTOS QUÍMICOS NO CONTROLE DA PODRIDÃO PARDA EM POMARES DE PESSEGUEIRO

Um experimento com controle biológico em campo foi conduzido no município da Lapa-PR, para o controle de Monilinia fructicola em pessegueiro cultivar BR-1. Agentes biológicos previamente selecionados de campo de produção foram comparados com o fungicida iprodione e com fosfitos de Ca e de K. O experimento contou com 10 tratamentos: seis fungos antagonistas (F1, F2 e F4 – Trichothecium roseum; F9 – Penicillium sp; F3 – Cephalosporium sp; F13 – Alternaria sp), dois fosfitos (Ca e K), iprodione e testemunha. A avaliação foi feita por incidência no campo e na pós-colheita, sendo os tratamentos aplicados por pulverização e imersão, respectivamente. Em relação à incidência em campo, destacaram-se o iprodione e, na seqüência, os fosfitos de K, com 71,4%, e de Ca, com 64,2% de controle. Entre os antagonistas, destacaram-se F3, F1 e F2, com nível de controle superior a 64% em relação à testemunha. Após a colheita, os frutos foram mantidos à temperatura ambiente e avaliados no terceiro dia de exposição pela incidência da doença. Nesse momento, foi observada uma baixa eficiência entre os tratamentos, somente destacando-se o fungicida. Também foram avaliados o peso médio dos frutos, diâmetro, firmeza da polpa e sólidos solúveis totais, não havendo diferença estatística entre os tratamentos. Paralelamente às avaliações de campo, frutos não pulverizados foram colhidos para receberem em pós-colheita os mesmos tratamentos executados no campo. Estes foram feridos, imersos nos tratamentos e avaliados pela incidência da doença no segundo e quarto dias de exposição no ambiente. Iprodione, fosfito de Ca e F1 exerceram o maior controle das infecções latentes. Nas áreas feridas, destacaram-se iprodione, F2 e F4.

Phytotoxicity assessment of diclofenac and its phototransformation products

The occurrence of pharmaceuticals in the environment is an emerging issue. Several studies observed that the non-steroidal anti-inflammatory drug diclofenac is ubiquitously present in most of the surveyed surface waters, worldwide. Phototransformation of diclofenac was reported from laboratory assays as well as in natural water systems, raising the question of possible adverse effects of the phototransformation products of diclofenac to aquatic organisms. In this study the phytotoxicity of diclofenac exposed to natural sunlight was evaluated using synchronized cultures of the unicellular chlorophyte Scenedesmus vacuolatus. Diclofenac dissolved in ultra-pure water at 50 mg L(-1) was exposed to natural midsummer sunlight for a maximum of 145 h. Twice a day subsamples were taken for chromatography and parallel phytotoxicity assessment. Inhibition of algal reproduction of the initial diclofenac solution was in the mg L(-1) range indicating no specific toxicity of diclofenac towards S. vacuolatus. Fast degradation of diclofenac was observed with half lives between 3.3 and 6.4 h during the first and the third day of exposure, respectively. Phytotoxicity increased after 3.5 h of exposure of diclofenac to sunlight and showed a maximum of sixfold enhanced toxicity after 53 h of exposure to sunlight. Several phototransformation products were found during the experiment. The time courses of the relative concentration of three transformation products significantly correlated with enhanced phytotoxicity during the experiment. This indicates a high toxicity potential of phototransformation products of diclofenac at concentration levels that may come close to environmental concentrations of residual diclofenac after degradation. We conclude that toxicity assessment of phototransformation products should be included in the risk assessment of pharmaceuticals in the environment.

The effect of a new chitin synthesis inhibitor, novaluron, on various developmental stages of Tribolium castaneum (Herbst)

Novaluron, a novel chitin synthesis inhibitor (CSI), was tested against the various developmental stages of Tribolium castaneum (Herbst), at concentrations ranging from 0.1 to 100 ppm. It did not kill T. castaneum adults at these concentrations, but at 1.0 ppm it caused total mortality of third-instar larvae. Novaluron did not affect the number of eggs laid by T. castaneum adults that were exposed to treated wheat flour, but it totally inhibited their hatching after the third day of exposure at a concentration of 1 ppm. On the first day after infestation novaluron did not totally prevent hatching, even at the highest tested concentration of 100 ppm. The time needed to restore egg hatchability after adults were transferred to untreated flour depended on the concentration of novaluron used in the treatment. When the adults of T. castaneum were exposed to novaluron-treated whole wheat grains (at 1 ppm), similar effects to those of the treated flour at the same concentration were observed: egg hatching was drastically reduced. The effect of uptake via contact of adults with novaluron-treated surfaces was examined by exposing them to a mixture of untreated flour and 10% novaluron-treated sand at a concentration of 10 ppm, and only 1% of the eggs hatched after 18 days of exposure. When adults were removed from treated surfaces of Petri dishes to untreated flour, the hatching rate was again reduced by 100%. It was concluded that the contact penetration of novaluron into T. castaneum adults prevents the hatching of eggs subsequently laid.

The effects of potassium and rubidium hydroxide on the alkali–silica reaction

Expansion of mortar specimens prepared with an aggregate of mylonite from the Santa Rosa mylonite zone in southern California was studied to investigate the effect of different alkali ions on the alkali–silica reaction in concrete. The expansion tests indicate that mortar has a greater expansion when subjected to a sodium hydroxide bath than in a sodium–potassium–rubidium hydroxide bath. Electron probe microanalysis (EPMA) of mortar bars at early ages show that rubidium ions, used as tracer, were present throughout the sample by the third day of exposure. The analysis also shows a high concentration of rubidium in silica gel from mortar bars exposed to bath solutions containing rubidium. The results suggest that expansion of mortar bars using ASTM C 1260 does not depend on the diffusion of alkali ions. The results indicate that the expansion of alkali–silica gel depends on the type of alkali ions present. Alkali–silica gel containing rubidium shows a lower concentration of calcium, suggesting competition for the same sites.

Effects of Cadmium on Chlorophyll Content, Photochemical Efficiency, and Photosynthetic Intensity of Canna indica Linn.

ABSTRACT The effects of cadmium (Cd2+) on growth status, chlorophyll (Chl) content, photochemical efficiency, and photosynthetic intensity were studied on Canna indica Linn. Plant specimens that were produced from a constructed wetland and precultivated hydroponically in 20 L of 1/10 Hoagland solution under greenhouse conditions for 1 week were exposed to cadmium in concentrations of 0, 0.4, 0.8, 1.6 and 3.2 mg L―1 Cd2+, respectively. The results show that leaves were injured in the Cd2+ solution by the third day of exposure and the injury became more serious with an increase in the applied heavy metal. Under 3.2 mg L―1 Cd2+ treatment, growth retardation, the decrease of chlorophyll content from 0.70 to 0.43 mg g―1 FW, and a decrease in Chl a/b ratio from 2.0 to 1.2 were observed. Chl a was more sensitive than Chl b to Cd2+ stress. The decrease was the same with photochemical efficiency. Photosynthetic intensity decreased by 13.3% from 1.5×104 μmol m―2s―1 CO2 in control to 1.3×104 μmol m―2s―1 CO2 in the treatment of 3.2 mg L―1. Because Canna species are used in heavy metal phytoremediation, these results show that C. indica can tolerate 0.4 to 0.8 mg L―1 Cd2+. Therefore, it is a potential species for phytoremediation of cadmium with some limitations only at higher concentrations.

Comparative Sensitivity of Seven Marine Microalgae to Cumulative Exposure to Ultraviolet-B Radiation with Daily Increasing Doses

A comparative study of the relative sensitivity to ultraviolet radiation (UVR) among seven marine microalgae from the classes Cyanophyceae (Synechococcus sp.), Rhodophyceae (Porphyridium cruentum), Cryptophyceae (Rhodomonas salina), Prymnesiophyceae (Isochrysis galbana), Bacillariophyceae (Chaetoceros sp.), Chlorophyceae (Dunaliella salina), and Prasinophyceae (Tetraselmis suecica), was carried out. Daily exposure for 4 h over a 4-day period with daily increasing doses of UVR (mainly ultraviolet-B, UV-B) led to a continuous decrease in the effective quantum yield (φPSII) in Rhodomonas salina, Chaetoceros sp. and Isochrysis galbana. In contrast, Tetraselmis suecica did not show a significant inhibition over the 4-day period of exposure. Synechococcus sp., Dunaliella salina and Porphyridium cruentum were able to restore, at least partially, the values of φPSII during the recovery period (white light only, 20 h), even though a significant decrease in φPSII was also measured in these algae after each exposure. Relative inhibition, defined here as the decrease in φPSII after exposure as compared to the value before exposure and normalised to the latter value (RI = ∆φ PSII /φ PSII ), was fitted against UV-B irradiance according to an equation of the type y = 1 ‐ (a x) c e -bx , where y is RI, x is the UV-B irradiance (W m ‐2 ), and a, b and c are constants determined from the fit, using unweighted and weighted irradiances. From the fit, the UV-B irradiance for 50% relative inhibition of φ PSII (RI = 0.5, E 50 [W m ‐2 ]), was calculated for each alga. Rhodomonas salina and Isochrysis galbana exhibited the lowest values of E50 (0.21 and 0.25, respectively, for unweighted irradiance), being the most sensitive algae to UV-B, while the highest value was calculated for Dunaliella salina (0.47 for unweighted irradiance). The E50 could not be calculated for Tetraselmis suecica since this alga showed an RI maximum of 0.1, and was therefore considered as a species which was highly tolerant of UV-B. A rise in optical density of each algal cell suspension at 680 and 750 nm, as well as in chlorophyll a content determined from methanolic extracts, was evident over the experimental period in all of the species except for Isochrysis galbana and Rhodomonas salina. In these two algae, in contrast to the other species, there was a notable increase in the carotenoid/chlorophyll a ratio, although this had negligible effects for the protection against deleterious UV-B. The phycobilin/chlorophyll a ratio showed a gradual decrease with time in the three phycobilin-containing algae, but it was particularly evident in R. salina after the third day of exposure.

Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC 6803.

We characterized the photosynthetic properties of the pmgA mutant of Synechocystis PCC 6803, which cannot change its photosystem stoichiometry under a high-light condition (200 micromol x m(-2) x s(-1)), in order to clarify the physiological significance of the regulation of photosystem stoichiometry. We found that (1) PSII activity was inhibited more in wild-type cells on the first day under the high-light conditions than in mutant cells. (2) The growth of the mutants following the initial imposition of high light was faster than that of wild-type cells. (3) However, growth was severely inhibited in the mutants after the third day of exposure to high light. (4) The growth inhibition in the mutants under the extended high-light conditions was reversed by the addition of sublethal concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which seemed to mimic photoinhibition of PSII. These results suggest that the main role of adjusting the photosystem stoichiometry with respect to light intensity is not to maintain efficient photosynthesis, but to down regulate electron transfer. Failure to down regulate electron flow leads to cell death under prolonged exposure to high light in this cyanobacterium.

Regulation of human placental lactogen expression by 1,25- dihydroxyvitamin D3

The human placenta synthesizes 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and expresses the vitamin D receptor (VDR), but the roles of 1,25-(OH)2D3 and the VDR in placental physiology are poorly understood. In this study, we have demonstrated that 1,25-(OH)2D3 stimulates the synthesis and release of human placental lactogen (hPL), one of the major secretory products of syncytiotrophoblast cells. Enzymatically dispersed trophoblast cells from term placentas exposed continuously to 1,25-(OH)2D3 (0.1, 6, and 37 microM) for 5 days released significantly more hPL than control cells after the third day of exposure. On days 4 and 5, the amounts of hPL released by cells exposed to 1,25-(OH)2D3 were 2.54- and 4.14-fold that of control cells (P < 0.001 in each instance). The stimulation by 1,25-(OH)2D3 was dose dependent and was accompanied by stimulation of hPL messenger RNA levels. Transient transfection studies of BeWo choriocarcinoma cells transfected with hPL promoter constructs coupled to the chloramphenicol acetyltransferase reporter gene indicated that the stimulation of hPL expression is due at least in part to stimulation of hPL gene expression. Deletion analysis studies of the hPL promoter indicated that a region between -500 to -1200 basepairs is necessary for 1,25-(OH)2D3 responsiveness. Analysis of this region shows a consensus vitamin D response element (VDRE) DNA-binding site of a direct repeat motif separated by three bases. Ligation of this placental VDRE site into a heterologous chloramphenicol acetyltransferase vector caused 1,25-(OH)2D3 responsiveness. Moreover, mobility shift assays demonstrated binding of VDR to placental VDRE. These results indicate that 1,25-(OH)2D3 stimulates the synthesis and release of hPL by a mechanism involving hPL gene transcription and support a role for vitamin D and the VDR in placental function.

Regulation of human placental lactogen expression by 1,25-dihydroxyvitamin D3.

The human placenta synthesizes 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and expresses the vitamin D receptor (VDR), but the roles of 1,25-(OH)2D3 and the VDR in placental physiology are poorly understood. In this study, we have demonstrated that 1,25-(OH)2D3 stimulates the synthesis and release of human placental lactogen (hPL), one of the major secretory products of syncytiotrophoblast cells. Enzymatically dispersed trophoblast cells from term placentas exposed continuously to 1,25-(OH)2D3 (0.1, 6, and 37 microM) for 5 days released significantly more hPL than control cells after the third day of exposure. On days 4 and 5, the amounts of hPL released by cells exposed to 1,25-(OH)2D3 were 2.54- and 4.14-fold that of control cells (P < 0.001 in each instance). The stimulation by 1,25-(OH)2D3 was dose dependent and was accompanied by stimulation of hPL messenger RNA levels. Transient transfection studies of BeWo choriocarcinoma cells transfected with hPL promoter constructs coupled to the chloramphenicol acetyltransferase reporter gene indicated that the stimulation of hPL expression is due at least in part to stimulation of hPL gene expression. Deletion analysis studies of the hPL promoter indicated that a region between -500 to -1200 basepairs is necessary for 1,25-(OH)2D3 responsiveness. Analysis of this region shows a consensus vitamin D response element (VDRE) DNA-binding site of a direct repeat motif separated by three bases. Ligation of this placental VDRE site into a heterologous chloramphenicol acetyltransferase vector caused 1,25-(OH)2D3 responsiveness. Moreover, mobility shift assays demonstrated binding of VDR to placental VDRE. These results indicate that 1,25-(OH)2D3 stimulates the synthesis and release of hPL by a mechanism involving hPL gene transcription and support a role for vitamin D and the VDR in placental function.

The effect of surgical glove powder on proliferation of human skin fibroblast and monocyte/macrophage.

The effect of surgical glove powders (Biosorb, Keoflo, and CaCO3) and Hydrocote (powder-free lubricating agent, Biogel) was examined on human skin fibroblasts and monocyte/macrophage cell lines (U937 and HL-60). Glove powders (0.1-100 micrograms/ml) in the presence of 10% fetal bovine serum (FBS) had no significant effect on the rate of 3H-thymidine uptake and proliferation of these cells after 48 h and 7 days of exposure, respectively. However, they inhibited HL-60 growth after 10 days, and Biosorb and CaCO3 inhibited U937 after 10-21 days of exposure compared with control. In the presence of low serum (0.5%), Biosorb, but not Keoflo, CaCO3, and Hydrocote, inhibited HL-60 cells after the third day of exposure (p < 0.05), whereas they were without any effect on U937 cells. Further incubation resulted in a significant decrease in cell density in all treatments, as well as controls, because of cell death. In the presence of 2% serum, glove powder-treated HL-60 significantly increased in cell numbers during the first 3 days, and the cells became stationary thereafter, whereas Keoflo and CaCO3-stimulated U937 reached a maximal by 9 days of treatment. Coculturing of fibroblasts directly with macrophages (0.4-5 x 10(5) cells per dish) or incubation with macrophage culture-conditioned media (CCM) stimulated quiescent fibroblast growth equal to that induced by 10% and 0.5% serum, respectively (p < 0.05). However, incubation of fibroblasts with glove powder-treated HL-60 CCM (except CaCO3) inhibited (p < 0.05) and CCM from Biosorb-treated U937 stimulated (p < 0.05) fibroblast proliferation. The CCM from glove powder-treated HL-60 and U937 did not have any significant effect on the rate of 3H-thymidine incorporation into fibroblasts compared with controls. The present observations suggest that glove powder action on fibroblast and macrophage growth in vitro depends on both the serum concentration of the culture medium and the length of exposure. The results imply that glove powders may have an adverse effect in vivo by directly influencing the biologic activity of macrophages, as well as other cell types, leading to alterations in the early phases of wound healing.

Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: stimulation of annexin I expression by dexamethasone.

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 +/- 33% increase comparing untreated cells and cells treated for 10 days with 5 x 10(-7) M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10(-10) M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.