Culture In Presence Research Articles

Developmental regulation of DUOX1 expression and function in human fetal lung epithelial cells

The purpose of this study was to determine the expression and cellular functions of the epithelial NADPH oxidase DUOX1 during alveolar type II cell development. When human fetal lung cells (gestational age 11-22 wk) were cultured to confluency on permeable filters, exposure of cells to a hormone mixture (dexamethasone, 8-Br-cAMP, and IBMX, together referred to as DCI) resulted in differentiation of cells into a mature type II phenotype as assessed by expression of lamellar bodies, surfactant proteins, and transepithelial electrical parameters. After 6 days in culture in presence of DCI, transepithelial resistance (2,616 +/- 529 Omega.cm(2)) and potential (-8.5 +/- 0.6 mV) indicated epithelial polarization. At the same time, treatment with DCI significantly increased the mRNA expression of DUOX1 ( approximately 21-fold), its maturation factor DUOXA1 ( approximately 12-fold), as well as DUOX protein ( approximately 12-fold), which was localized near the apical cell pole in confluent cultures. For comparison, in fetal lung specimens, DUOX protein was not detectable at up to 27 wk of gestational age but was strongly upregulated after 32 wk. Function of DUOX1 was assessed by measuring H(2)O(2) and acid production. Rates of H(2)O(2) production were increased by DCI treatment and blocked by small interfering RNA directed against DUOX1 or by diphenylene iodonium. DCI-treated cultures also showed increased intracellular acid production and acid release into the mucosal medium, and acid production was largely blocked by knockdown of DUOX1 mRNA. These data establish the regulated expression of DUOX1 during alveolar maturation, and indicate DUOX1 in alveolar H(2)O(2) and acid secretion by differentiated type II cells.

Batch Nitrifying Cultures in Presence of Mixtures of Benzene, Toluene, and m-Xylene

Benzene, toluene, and m-xylene compounds in individual (5.0 ± 0.5 mg C l−1) and mixed solutions (2.5 ± 0.2 mg C l−1 for each one) in nitrifying batch cultures induced a decrease in the specific rates of NH4 + consumption (81 ± 6%) and NO3 − production (39-79%). However, after 24 h, ammonium consumption efficiency and conversion of consumed NH4 +-N into NO3 −-N were close to 100% and there was no significant accumulation of nitrite in the medium. After 24 h, no aromatic intermediate was detected in the cultures and 50% of the mixed compounds was converted to acetate. The following order of biotransformation was found: m-xylene > toluene > benzene. Transformation rate of m-xylene was 0.051 ± 0.005 g C (g protein-N h)−1 in individual and mixed solutions. When m-xylene was added, benzene was oxidized at a faster rate (0.051 ± 0.005 g C (g protein-N h)−1) whereas toluene at a slower rate (0.012 ± 0.002 g C (g protein-N h)−1).

Solubilization of inorganic phosphates by fungi isolated from agriculture soil

Most agricultural soils contain large reserves of phosphorus (P), a considerable part of which accumulates as a consequence of regular applications of P fertilizers. However, a greater part of soil phosphorus, approximately 95‐99% is present in the form of insoluble phosphates and hence cannot be utilized by the plants. In the present study fungal strains isolated from agriculture soil, having potential to solubilize insoluble inorganic phosphates were characterized. Two fungal isolates were tested for their tricalcium phosphate (TCP) solubilization efficiency in both solid and liquid medium. Isolates were identified as Aspergillus sp. and Penicillium sp. depending upon their colony morphology and microscopic studies. Phosphate solubilization was related to pH decrease caused by growth of fungus in medium containing glucose as carbon source. Aspergillus sp. solubilized 480 �g/ml of phosphorus, while Penicillium sp. solubilized 275 �g/ml of phosphorus from 0.5% tricalcium phosphate after 4 and 3 days of growth respectively. Both the strains show diverse levels of phosphate solubilization activity in liquid broth culture in presence of various carbon and nitrogen sources. Drop in pH during growth was more prominent in absence of TCP in the liquid medium. This indicates that absence of soluble P in media induces the acid production. Phosphate solubilizing microorganisms convert insoluble phosphates into soluble forms generally through the process of acidification, chelation and exchange reactions. Thus such microorganisms may not only compensate for higher cost of manufacturing fertilizers in industry but also mobilizes the fertilizers added to soil.

Solubilization of inorganic phosphates by fungi isolated from agriculture soil

Most agricultural soils contain large reserves of phosphorus (P), a considerable part of which accumulates as a consequence of regular applications of P fertilizers. However, a greater part of soil phosphorus, approximately 95‐99% is present in the form of insoluble phosphates and hence cannot be utilized by the plants. In the present study fungal strains isolated from agriculture soil, having potential to solubilize insoluble inorganic phosphates were characterized. Two fungal isolates were tested for their tricalcium phosphate (TCP) solubilization efficiency in both solid and liquid medium. Isolates were identified as Aspergillus sp. and Penicillium sp. depending upon their colony morphology and microscopic studies. Phosphate solubilization was related to pH decrease caused by growth of fungus in medium containing glucose as carbon source. Aspergillus sp. solubilized 480 �g/ml of phosphorus, while Penicillium sp. solubilized 275 �g/ml of phosphorus from 0.5% tricalcium phosphate after 4 and 3 days of growth respectively. Both the strains show diverse levels of phosphate solubilization activity in liquid broth culture in presence of various carbon and nitrogen sources. Drop in pH during growth was more prominent in absence of TCP in the liquid medium. This indicates that absence of soluble P in media induces the acid production. Phosphate solubilizing microorganisms convert insoluble phosphates into soluble forms generally through the process of acidification, chelation and exchange reactions. Thus such microorganisms may not only compensate for higher cost of manufacturing fertilizers in industry but also mobilizes the fertilizers added to soil.

Poly- β-hydroxybutyrate accumulation in Nostoc muscorum: Effects of metabolic inhibitors

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.

Plantlet regeneration through leaf callus culture in sugarcane

The young meristematic leaf sheath expiants of sugarcane genotype S-3807/99 were aseptically inoculated on agar (7.5 g/1) gelled Murashige and Skoog’ (MS) medium containing 20 g/1 sucrose andNAA, IBA and 2,4-D for callus formation. Maximum 67.3% expiants showed callus initiation within 10–14 days at 4.0 mg/1 2,4-D. The actively growing calli were transferred on agar solidified MS medium fortified with different concentrations of BAP, Kn and NAA. Shoot regeneration was recorded in 58.3% callus cultures in presence of BAP and Kn (1.0 mg/1 each) along with NAA (0.5 mg/1). Maximum shoot multiplication alongwith vigorous shoot growth was observed on medium containing BAP, Kn and GA1(1.0 mg/1 each) along with NAA (0.5 mg/1). Well grown shoots were rooted on 1/2 strength MS liquid medium containing NAA (5.0 mg/ 1) and sucrose (50 g/1). The rooted plantlcts were hardened in polythene bags containing soil mixture under green house condition where about 85% survival was recorded. The hardened plantlets (somaclones) were transplanted in the field for evaluation.

Étude de l’effet des oligomères procyanidoliques sur la fibrillogénèse de la cornée

We showed in a previous study that procyanidolic oligomers (PCOs) from grape seeds effectively protect corneal stroma against degradation by bacterial collagenase. Here we report the study of the effect of PCOs on protein and collagen composition of cornea and on the biosynthesis of corneal collagens. Bovine corneas were used in explant cultures. We quantitatively determined total proteins and collagen as well as the incorporation of 3H-proline in separated collagen types. Collagens type I, V, and VI were separated and quantitated. In order to understand some of the results obtained, we studied the interaction of PCO with collagen type I separately. In the absence of PCOs, collagen typing and 3H-proline incorporation yielded the expected results for a normal cornea, with the usual proportion of the three major types of collagens. In presence of PCOs at 1 mg/ml and after 24 h incubation, total proteins and collagens decreased, as did papain-extractable collagens. Proteins in the final residue solubilized in 1 M KOH-80% v/v aqueous ethanol increased. The proportion of the three principal collagens was also modified: type I became preponderant, and the proportions of the two others (type V and VI) decreased. The study of the interaction of collagen type I with PCOs showed that 30% of total PCOs do not interact with collagen, 20% interact reversibly, and 50% of PCOs are strongly and irreversibly fixed. This strongly fixed fraction could not be separated from collagen by either column chromatography or collagenase or KOH in aqueous ethanol. Bovine corneas in explant cultures in presence of PCOs undergo a modification of their protein and collagen content, with a concurrent modification of the proportion of collagens types I, V, and VI. Collagen type I predominates, and the two other types decrease. Study of the collagen type I-PCO interaction showed that roughly 50% of PCOs become irreversibly fixed to collagen, resisting collagenase or other methods of separation. This strong PCO-collagen type I interaction explains the decrease in solubility.

Valproic Acid Accelerates Cell Cycle Progression of Hematopoietic Stem Cells Via the Activation of GSK3beta-Dependent Signaling Pathways.

Histone deacetylase inhibitors (HDI) have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts either alone, or in combination with differentiating agents such as all-trans retinoic acid (ATRA). We have previously reported favorable effects of the potent HDI valproic acid (VPA) in combination with ATRA in a small subset of patients with advanced acute myeloid leukemia (AML) leading to blast cell reduction and improvement of hemoglobin. This effect was accompanied by hypergranulocytosis most likely due to an enhancement of non-leukemic myelopoiesis and suppression of malignant hematopoiesis rather than enforced differentiation of leukemic cells. These data prompted us to investigate the impact of VPA on normal hematopoietic stem cells (HSC).Differentiation of cord blood-derived, purified CD34+ cells was assessed by FACS analysis after a 7-days suspension culture in presence of early acting cytokines and 30–150μg/mL VPA. VPA prevented differentation of CD34+ cells in a dose-dependent manner: concomitant with an increase of CD34+ cells from 17 to 47%, the proportion of monocytic CD14+ cells decreased from 27 to 3% (n=3). In addition, VPA induced a 30-fold amplification of CD34+ bone marrow (BM) cells within 10 days as determined by colony assays (n=3). To evaluate the functional capacity of VPA-treated HSC, murine Sca1+/lin−s cells were harvested from colony assays and replated. VPA treatment allowed up to four cycles of replating in contrast to VPA-naïve control cells. Further analysis demonstrated that the stimulatory effect of VPA on the in vitro growth and colony formation capacity of HSC was mainly due to accelerated cell cycle progression. VPA strongly increased the proportion of cells in S phase compared to untreated controls (38 vs. 17%, resp.), as detected by propidium iodid staining and BRDU incorporation as well as reduced expression of the CDK-inhibitor p21cip-1/waf using murine HSC after 7 days of culture. Downregulation of p21cip-1/waf was confirmed in CD34+ BM cells showing maximum inhibition after 48 hours of VPA treatment and no recovery thereafter. Recent results indicate that VPA exerts inhibitory activity on GSK3beta by phosphorylation on Ser-9 and stimulates Akt in human neuroblastoma cells. GSK3beta is an effector of the Wnt-signaling pathway located upstream of beta-catenin. Wnt-signaling can directly stimulate the proliferation of HSC, expand the HSC pool and lead to upregulation of HoxB4. Here we show that VPA increased the inhibition-associated phosphorylation of GSK3beta on Ser-9 in human CD34+ BM cells after 48 hours as well as in murine Sca1+/lin− cells after 7 days. Exposure to VPA enhanced beta-catenin and Akt activity not only in CD34+ HSC but also in KG-1 and TF-1 cells with maximum activation after 48 hours of VPA stimulation. Moreover, VPA lead to an 8-fold increase of the HoxB4 level in CD34+ BM cells as determined by real time PCR at 48 hours.In conclusion, we show that VPA i.) expands HSC as assesed by phenotype and function; ii.) accelerates cell cycle progression of HSC accompanied by the down-regulation of p21cip-1waf; iii.) activates the GSK3beta depending beta-catenin pathway and Akt and iv.) up-regulates HoxB4. Our data strongly suggest that VPA is able to influence some of the signaling pathway considered relevant for proliferation and self-renewal which might request reconsideration of their employment for the treatment of AML.

Restorative potential of dopaminergic grafts in presence of antioxidants in rat model of Parkinson's disease

Free radical mediated damage has been reported to contribute significantly towards low survival (5–10%) of grafted dopaminergic neurons, post transplantation. In the present study, an attempt has been made to explore the neuroprotective potential of the combination of two major antioxidants ascorbic acid (AA) and glutathione (GSH) on ventral mesencephalic cells (VMC) and nigral dopamine (DA) neurons when co-transplanted together with VMC in rat model of Parkinson's disease (PD). GSH and AA have been reported to act co-operatively in the conditions of oxidative stress thereby helping in maintaining the cellular GSH/GSSG redox status. Functional recovery was assessed 12 weeks post transplantation, where a significant restoration ( p < 0.001) in d-amphetamine induced circling behavior (62%), spontaneous locomotor activity (SLA; 64%), dopamine–D 2 receptor binding (63%), dopamine (65%) and 3,4-dihydroxy phenyl acetic acid (DOPAC) level (64%) was observed in co-transplanted animals as compared to lesioned and VMC alone grafted rats. VMC and GSH + AA co-transplanted animals exhibited a significantly higher surviving TH-immunoreactive (TH-ir) neurons number ( p < 0.01), TH-ir fibers outgrowth ( p < 0.05) in striatal graft and TH-ir neurons in substantia nigra pars compacta (SNpc) ( p < 0.01), as compared to VMC alone transplanted rats. An attempt was made to further confirm our in vivo observations through in vitro experiments where following in vitro exposure to 6-OHDA, a higher cell survival ( p < 0.01), TH-ir cell counts ( p < 0.001) and DA and DOPAC levels ( p < 0.01) were also observed in 8-day-old VMC culture in presence of GSH + AA as compared to VMC cultured in absence of antioxidants. The results suggest that GSH + AA when co-transplanted with VMC provide higher restoration probably by increasing the survival of grafted VMC and simultaneously supporting nigral TH-immunopositive neurons in rat model of PD.

INFLUENCE OF POLYAMINES ON THE SPORULATION OF GRATELOUPIA (HALYMENIACEAE, RHODOPHYTA)1

Polyamines (PAs) such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are ubiquitous aliphatic amines involved in widely varying physiological behavior, but particularly they are actively involved in cell growth, division, and differentiation during reproductive events in plants. The contents of PUT, SPD, and SPM in infertile and fertile thalli of the red macroalga Grateloupia sp. were compared, and the results revealed a significant decrease in quantity from infertile to fertile status. At the enzymatic level, l‐ornithine decarboxylase (ODC) was mainly detected, and l‐arginine decarboxylase activity was not diminished by the inhibition of ODC. The maximum enzymatic activities, within the range of activities observed, correlated with the lower levels of polyamines in fertile thalli. In culture, SPM promoted the maturation of cystocarps to the eventual liberation of spores from aseptic fertile explants. PAs accumulated in cultivated explants as compared with noncultivated, but exogenous SPM addition further increased the endogenous SPM. The addition of berenyl, cordycepin, cyclohexylamine, dicyclohexylamine, and aurintricarboxylic acid blocked the synthesis in culture at the level of PUT, and partially at SPD and SPM synthesis, but the addition of SPM restored the levels of SPD and SPM as SPM accumulated, and they appeared to interconvert each other. The results obtained suggest that the culture in presence of SPM restored a deficient SPM situation in fertile explants, thus promoting sporulation.

ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8alpha(+) dendritic cells.

Interferon (IFN) consensus sequence-binding protein (ICSBP) is a transcription factor playing a critical role in the regulation of lineage commitment, especially in myeloid cell differentiation. In this study, we have characterized the phenotype and activation pattern of subsets of dendritic cells (DCs) in ICSBP−/− mice. Remarkably, the recently identified mouse IFN-producing cells (mIPCs) were absent in all lymphoid organs from ICSBP−/− mice, as revealed by lack of CD11clowB220+Ly6C+CD11b− cells. In parallel, CD11c+ cells isolated from ICSBP−/− spleens were unable to produce type I IFNs in response to viral stimulation. ICSBP−/− mice also displayed a marked reduction of the DC subset expressing the CD8α marker (CD8α+ DCs) in spleen, lymph nodes, and thymus. Moreover, ICSBP−/− CD8α+ DCs exhibited a markedly impaired phenotype when compared with WT DCs. They expressed very low levels of costimulatory molecules (intercellular adhesion molecule [ICAM]-1, CD40, CD80, CD86) and of the T cell area-homing chemokine receptor CCR7, whereas they showed higher levels of CCR2 and CCR6, as revealed by reverse transcription PCR. In addition, these cells were unable to undergo full phenotypic activation upon in vitro culture in presence of maturation stimuli such as lipopolysaccharide or poly (I:C), which paralleled with lack of Toll-like receptor (TLR)3 mRNA expression. Finally, cytokine expression pattern was also altered in ICSBP−/− DCs, as they did not express interleukin (IL)-12p40 or IL-15, but they displayed detectable IL-4 mRNA levels. On the whole, these results indicate that ICSBP is a crucial factor in the regulation of two possibly linked processes: (a) the development and activity of mIPCs, whose lack in ICSBP−/− mice may explain their high susceptibility to virus infections; (b) the generation and activation of CD8α+ DCs, whose impairment in ICSBP−/− mice can be responsible for the defective generation of a Th1 type of immune response.

Cell contact-dependent PMN HLA-DR and CD69 membrane expression induced by autologous mono-lymphocytes and cell lines.

Polymorphonuclear granulocytes (PMN) are commonly considered short-lived cells playing an efficient role in primary host defense via phagocytosis and release of cytotoxic compounds and inflammatory cytokines. Purified PMN do not express HLA-DR and CD69 molecules on cell surface, but they can be induced to do so by co-culture with peripheral blood derived mono-lymphocytes. De novo cell-surface expression of HLA-DR was also induced in PMN by co-culture with cell lines of lymphoid phenotype, but not with cell lines of myeloid phenotype. CD69 expression was not induced by co-culture with any of the cell lines used in the present study. In addition, we have observed induction of HLA-DR surface expression on PMN by culture in presence of culture supernatant of one of the cell lines of lymphoid origin, RPMI-8866. Quantitative analysis of HLA-DR and CD69 expression in stimulated PMN allowed us to divide PMN donors in two main groups, one with low expression and the other with high expression of the two molecules. HLA-DR surface expression was not altered by treatment with CHX and BFA, and RT-PCR analysis of total RNA from resting and stimulated PMN with RPMI-8866 supernatant did not detect the presence of any specific HLA-DR and CIITA transcript. Flow-cytometry and fluorescence microscopy analysis of resting PMN revealed the presence of HLA-DR molecules localized in intracellular vesicular-tubular structures. These data show that a reservoir of HLA-DR molecules is stored in the cytoplasm of human resting PMN and can be released to reach cell surface by a mobilization mechanism induced by cell surface interactions with selected cell types and sometimes with molecules released in culture supernatants.

Optimisation des conditions de culture pour l’enracinement in vitro de l’arganier ( Argania spinosa L.)

The root system produced of in vitro argan plantlets is of poor quality and not efficient for the transfer to out-door conditions. To overcome such problems, experimentation was undertaken where the effects of growth regulators, nitrogen, sugar, activated charcoal and coconut fiber were tested on root induction and elongation. Modified Murashige and Skoog with half strength salt was used as a basal medium. Root induction (85 %) with a mean of 16 roots per explant was obtained when shoots were grown, under dark conditions for 14 days, with a combination of two auxins (IBA and NNA), added at equal concentrations (5 mg·L –1). Secondary roots, 10 cm long, were initiated in 12 % of the cultures in presence of 5 g·L –1 activated charcoal. Further improvements in the growth of the primary and secondary roots were obtained when semi-solid medium was substituted with a substrate composed of coconut fibers (80 g) mixed with semi-solid medium (35 mL) and agar (2.5 g·L –1).

Production of Acinetobacter radioresistens lipase with repeated batch culture in presence of nonwoven fabric.

Cultivation of Acinetobacter radioresistens on n-hexadecane for lipase production was investigated with repeated batch culture in the presence of a hydrophobic nonwoven fabric. Lipase production followed the growth-associated model, and the repeated batch culture could achieve both high enzyme yield and increased volumetric productivity. The fabric was shown to be able to disperse n-hexadecane, to adsorb the unused hydrocarbon, and to retain bioemulsifiers excreted from the cells; therefore, it enhanced cell growth and, in turn, lipase production. In the repeated batch culture in the absence of the fabric, lipase yield and volumetric productivity were found to be 21 U/mL and 875 U/L. h, respectively. However, if the fabric was equipped in the fermentor, lipase yield and volumetric productivity increased to 30 U/mL and 2500 U/L. h, respectively. The lipase production profile could be further improved by raising the amount of nitrogen source and, as a result, a lipase yield of 54 U/mL and a volumetric productivity of 2250 U/L. h were obtained. In this study we assess the beneficial effects of nonwoven fabric on lipase production.

Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli

Stannous ion has been used in different sectors of human interest, such as in food industry and in health sciences. Much is known about stannous chloride (SnCl 2) toxicity, although, there is no general agreement regarding its genotoxicity. Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts have been used in popular medicine. We evaluated the influence of these crude extracts on the survival of the Escherichia coli wild type (AB 1157) strain submitted to SnCl 2 treatment. Reactive oxygen species (ROS) can be generated by a Fenton like reaction induced by SnCl 2. E. coli culture was treated simultaneously with SnCl 2 and a specific extract. Our results showed a reduction of the SnCl 2 effect on the survival of the cultures in presence of the crude extracts. The extract of M. ilicifolia showed the highest level of protection action against the SnCl 2 effect in comparison with the other extracts. This protector effect could due to the redox properties of these crude extracts. The compounds in the crude extracts could (i) chelate stannous ions, protecting them against the oxidation and avoiding the generation of ROS, (ii) be a scavenger of the ROS generated by the SnCl 2 oxidation and/or (iii) have oxidant compounds that could oxidise the stannous ions, abolishing or reducing the SnCl 2 effect.

Inhibition of nitric oxide synthase abrogates lipopolysaccharides-induced up-regulation of L-arginine uptake in rat alveolar macrophages.

It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides-(LPS)-induced up-regulation of L-arginine transport in rat alveolar macrophages (AM). AM were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]-L-arginine uptake or to isolate RNA for RT - PCR. Culture in presence of LPS (1 microg ml(-1), 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]-L-arginine uptake. The inducible NO synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) present alone during culture had only marginal effects on [3H]-L-arginine uptake. However, AMT present during culture additionally to LPS, suppressed LPS-induced nitrite accumulation and LPS-stimulated [3H]-L-arginine uptake in the same concentration-dependent manner. AMT present only for the last 30 min of the culture period had similar effects on [3H]-L-arginine uptake. AMT present only during the uptake period also inhibited LPS-stimulated [3H]-L-arginine uptake, but with lower potency. The inhibitory effect of AMT could not be opposed by the NO releasing compound DETA NONOate. LPS caused an up-regulation of the mRNA for the cationic amino acid transporter CAT-2B, and this effect was not affected by AMT. AMT (100 microM) did not affect L-arginine transport studied by electrophysiological techniques in Xenopus laevis oocytes expressing either the human cationic amino acid transporter hCAT-1 or hCAT-2B. In conclusion, iNOS inhibition in rat AM abolished LPS-activated L-arginine uptake. This effect appears to be caused by reduced flow of L-arginine through the iNOS pathway.

Quaternary ammonium compound stresses induce specific variations in fatty acid composition of Pseudomonas aeruginosa

The involvement of cell membrane fatty acids in resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds (QACs) stresses was investigated. The strain was grown in a medium with increasing concentrations of different biocides: two QACs, and two non-QACs. In the presence of two QACs only, the strain was able to grow with increasing concentrations. During cellular adaptation to QACs, the resistance to the same biocide increased. A principal component analysis was performed with whole of fatty acid compositions which highlighted a specific variation for the cultures in presence of QACs. These modifications gave evidence of the outer membrane involvement in cellular response to the presence of QACs.

Antitumor and antiviral activity of Colombian medicinal plant extracts.

Extracts of nine species of plants traditionally used in Colombia for the treatment of a variety of diseases were tested in vitro for their potential antitumor (cytotoxicity) and antiherpetic activity. MTT (Tetrazolium blue) and Neutral Red colorimetric assays were used to evaluate the reduction of viability of cell cultures in presence and absence of the extracts. MTT was also used to evaluate the effects of the extracts on the lytic activity of herpes simplex virus type 2 (HSV-2). The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration of the viral effect (EC50) for each extract were calculated by linear regression analysis. Extracts from Annona muricata, A. cherimolia and Rollinia membranacea, known for their cytotoxicity were used as positive controls. Likewise, acyclovir and heparin were used as positive controls of antiherpetic activity. Methanolic extract from Annona sp. on HEp-2 cells presented a CC50 value at 72 hr of 49.6x10(3)mg/ml. Neither of the other extracts examined showed a significant cytotoxicity. The aqueous extract from Beta vulgaris, the ethanol extract from Callisia grasilis and the methanol extract Annona sp. showed some antiherpetic activity with acceptable therapeutic indexes (the ratio of CC50 to EC50). These species are good candidates for further activity-monitored fractionation to identify active principles.

Id4 Expression Induces Apoptosis in Astrocytic Cultures and Is Down-regulated by Activation of the cAMP-Dependent Signal Transduction Pathway

The Id family of helix–loop–helix transcription factors has been implicated in the regulation of cellular differentiation in several different lineages. We have explored the potential regulatory role of the cyclic AMP-dependent signaling pathway on Id gene expression in astroglial primary cultures. We found that primary cultures of mouse forebrain astrocytes constitutively expressed the four known members of the Id gene family, Id1, Id2, Id3, and Id4. During culture in presence of serum for 4 weeks, the expression of Id4 was up-regulated. In these same cultures, treatment with dibutyryl-cyclic AMP, a cyclic AMP analogue known to promote astrocyte differentiation, dramatically and selectively decreased Id4 gene expression. This effect was detectable after short-term treatment and was maintained during long-term treatment. Forskolin and pentoxifylline, two other agents known to elevate intracellular cyclic AMP through different mechanisms, also potently decreased Id4 gene expression. Furthermore, overexpression of Id4 in an astrocyte-derived cell line induced cells to round up and die by apoptosis. These results indicate that the cyclic AMP pathway acts as an inhibitor of Id4 gene expression in astrocytes, identify a new function for Id4, and suggest that Id4 is strategically positioned in the chain of molecular events regulating astrocyte differentiation and apoptosis.

A stress-inducible rat liver endoplasmic reticulum protein, ERp29.

We have isolated, cDNA cloned and characterised a 29-kDa protein (ERp29), containing a C-terminal endoplasmic reticulum(ER)-retrieval signal, from the rat liver ER. ERp29 was induced to high levels in the rat hepatoma cells under metabolic stress conditions known to cause an aberrant accumulation of proteins in the ER [(e.g. culture in presence of the Ca2+ ionophore A23187, inhibitors of Ca2+-ATPase (thapsigargin), intracellular protein transport (brefeldin A), or protein N-glycosylation (tunicamycin)]. Experimental evidence of its localisation in the luminal compartment of the ER was obtained by topology studies including immunofluorescence microscopy, in vitro translation and proteinase protection assay. ERp29 constitutes about 0.1% of the rat hepatic microsomal proteins and is constitutively expressed in all rat tissues examined, as evident from northern blot analysis. In rat hepatoma cells ERp29 was found to be associated with the abundant molecular chaperone/stress protein BiP/GRP78 and this interaction was significantly enhanced after treatment with tunicamycin and A23187. Taken together, these results suggest that ERp29 is a member of the stress-response machinery of the ER.