Production of prostacyclin by different cell types of the goat ovary

A sensitive platelet aggregation-inhibition assay was used to quantitate the production of prostacyclin by different cell types of the goat ovary. The assay could detect as low as 0.16 ng in the test sample. Different cell types i.e. granulosa, theca and corpus luteum or the total ovarian homogenate were incubated at 37° C for 10 minutes with or without 0.2mM arachidonic acid. Rat aortic strips were incubated under similar conditions as a positive control. Under basal conditions the amount of prostacyclin produced by corpus luteum cells was higher compared to that by granulosa cells. When the precursor of prostaglandins (arachidonic acid) was provided the production markedly increased in corpus luteum, granulosa, and ovarian homogenate as well as in aortic strips. Theca cells did not produce detectable levels of prostacyclin even when the precursor was provided. Trapidil did not alter the basal but enhanced the archidonic acid-stimulated prostacyclin production in homogenate and granulosa cells with no further increase in corpus luteum cells. U-51605 decreased basal as well as arachidonic acid-stimulated prostacyclin production in all the cell types. The prostacyclin production in ovaries is compartmentalized suggesting a possible role in ovarian physiology.     

Phospholipase C activation by prostacyclin receptor agonist in cerebral microvascular smooth muscle cells

The mechanism through which iloprost permits cerebral vasodilation induced by specific stimuli is incompletely understood. Previous study suggests there might be interplay between the adenylyl cyclase and phospholipase C (PLC) systems. Coupling of the prostacyclin receptor with the PLC pathway system was investigated. Iloprost, a stable prostacyclin analog, was used as a prostacyclin receptor agonist. We investigated the effects of iloprost (10-12-10-6 M) on inositol 1,4,5-trisphosphate (IP3) production by piglet cerebrovascular smooth muscle cells in primary culture. Iloprost caused concentration- and time-dependent increases in IP3 production in control cells and in cells pretreated with LiCl (to prevent further IP3 metabolism). Iloprost treatment (10-12 M) of cerebrovascular smooth muscle cells, in the absence and presence of 20 mM LiCl, resulted in 2-fold and 4-fold increases in the formation of IP3, respectively. In contrast, 10-10 M to 10-6 M iloprost, either in the presence or absence of LiCl, induced moderate or no increase in IP3 formation. Iloprost (10-10-10-12 M) strongly stimulated diacylglycerol (DAG) generation, whereas higher concentrations (10-8 M) did not induce an increase. In conclusion, the results suggest that prostacyclin receptors on cerebromicrovascular smooth muscle can couple to PLC, generating the second messengers, IP3 and DAG.     

Synthesis and evaluation of N-acylsulfonamide and N-acylsulfonylurea prodrugs of a prostacyclin receptor agonist

N-Acylsulfonamide and N-acylsulfonylurea derivatives of the carboxylic acid prostacyclin receptor agonist 1 were synthesized and their potential as prodrug forms of the carboxylic acid was evaluated in vitro and in vivo. These compounds were converted to the active compound 1 by hepatic microsomes from rats, dogs, monkeys, and humans, and some of the compounds were shown to yield sustained plasma concentrations of 1 when they were orally administered to monkeys. These types of analogues, including NS-304 (2a), are potentially useful prodrugs of 1.     

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.     

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types.     

Tyrosine phosphorylation of the kappa -opioid receptor regulates agonist efficacy

To explore the role of highly conserved tyrosine residues in the putative cytoplasmic domains of the seven-transmembrane G protein-coupled opioid receptors, we expressed the rat kappa-opioid receptor (KOR) in Xenopus oocytes and then activated the intrinsic insulin receptor tyrosine kinase. KOR activation by the agonist produced a strong increase in potassium current through coexpressed G protein-gated inwardly rectifying potassium channels (K(IR)3). Brief pretreatment with insulin caused a 60% potentiation of the KOR-activated response. The insulin-induced increase in kappa-opioid response was blocked by the tyrosine kinase inhibitor genistein. In contrast, insulin had no effect on the basal activity of K(IR)3, suggesting that KOR is the target of the tyrosine kinase cascade. Mutation of tyrosine residues to phenylalanines in either the first or second intracellular loop of KOR to produce KOR(Y87F) and KOR(Y157F) had no effect on either the potency or maximal effect of. However, neither KOR(Y87F)- nor KOR(Y157F)-mediated responses were potentiated by insulin treatment. Insulin pretreatment shifted the dose-response curve for activation of KOR by increasing the maximal response without changing the EC(50) value for. These results suggest that insulin increases the efficacy of KOR activation by phosphorylating two tyrosine residues in the first and second intracellular loops of the receptor. Thus, tyrosine phosphorylation may provide an important mechanism for modulation of G protein-coupled receptor signaling.     

Galectin-1 receptors in different cell types

Summary Galectins are a family of animal lectins defined by two properties: shared amino acid sequences in their carbohydrate-recognizing domain, and -galactoside affinity. A wide variety of biological phenomena are related to galectins, i.e., development, differentiation, morphogenesis, tumor metastasis, apoptosis, RNA splicing, and immunoregulatory function. In this review, we will focus on galectin-1 receptors, and some of the mechanisms by which this lectin affects different cell types. Several galectin-1 receptors are discussed such as CD45, CD7, CD43, CD2, CD3, CD4, CD107, CEA, actin, extracellular matrix proteins such as laminin and fibronectin, glycosaminoglycans, integrins, a -lactosamine glycolipid, GM1 ganglioside, polypeptide HBGp82, glycoprotein 90 K/MAC-2BP, CA125 cancer antigen, and pre-B cell receptor.This revised version was published online in April 2005. In the previous version the name of the last author was missing.     

Stereochemistry of glutamate receptor agonist efficacy: engineering a dual-specificity AMPA/kainate receptor

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.     

Negative dielectrophoretic patterning with different cell types

In this paper, a novel method for patterning different cell types based on negative dielectrophoresis (n-DEP), without any special pretreatment of a culture slide, has been described. An interdigitated array (IDA) electrode with four independent microelectrode subunits was fabricated with indium-tin-oxide (ITO) and used as a template to form cellular micropatterns. A suspension of C2C12 cells was introduced into the patterning device between the upper slide and the bottom IDA. In the present system, the n-DEP force is induced by applying an ac voltage (typically 12V(pp), 1MHz) to direct cells toward a weaker region of electric field strength. The cells aligned above one of the bands of IDA within 1min since the aligned areas on the slide were regions with the lower electric field. The application of an ac voltage for 5min allows the cells to adsorb onto the cell culture slide. After removing excess cells, the second cell type was patterned in lines using the same method as with the first set of cells. Periodic and alternate cell lines incorporating two cell types were also fabricated by changing the ac voltage mode. A second cell type was introduced into the device and guided to other areas to form a different pattern. The described system enables two cell types to be patterned in 15min. The patterning method provides a novel tool for use in fundamentals studies of cell biology based on cell-cell interactions between different cell types.     

Factors affecting nitrogen loss in experimental wetlands with different hydrologic loads

Constructed or restored wetlands have great potential for reducing nonpoint source contamination of surface and ground waters by agricultural chemical contaminants. The work reported here combines field and experimental studies of factors affecting nitrogen loss in the Des Plaines River Experimental Wetlands, northeastern Illinois, USA. These wetlands receive approximately 5–36 cm/week of pumped river water with significant but seasonally variable loads of nitrate and organic nitrogen. On an annual basis, the wetlands removed 78–95% of the nitrate and 54–75% of the total nitrogen received. At the low hydrologic loading rate, organic nitrogen exports approximately equalled imports. However at the higher hydrologic loading rate, the wetlands exported 22–31% more organic nitrogen than received. Seasonal variation in nitrate and organic nitrogen loads had significant effects on the effectiveness of the wetlands as sinks for total nitrogen. The wetlands were nitrogen sinks during periods of the nitrate loading and nitrogen sources during periods of low nitrate loading. Experimental studies demonstrated the effects of nitrate concentration, temperature, and location on rates of nitrate loss. Results suggest that nitrite loading rates might influence not only nitrate loss rates but also loss rate coefficients.     

Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.

Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1-subunit of the rat brain GABAA receptor. We separately co-expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1-Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60- to 200-fold by these mutations in alpha-subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha-subunits with the agonist/antagonist binding site, and involvement of N-terminal portions of the extracellular domains of all subunits in the gating of the channel.     

The potency of acyclovir can be markedly different in different cell types

Acyclovir is an acyclic guanine analog with a considerable activity against herpes simplex viruses. We studied the antiherpetic activity of acyclovir in macrophages and fibroblast cell lines. Utilising a plaque reduction assay we found that acyclovir potently inhibited the HSV-1 replication in macrophages (EC50) = 0.0025 microM) compared to Vero (EC50 = 8.5 microM) and MRC-5 (EC50 = 3.3 microM) cells. The cytotoxicity of acyclovir was not detected at concentrations < or = 20 microM, thus the selective index in macrophages was >8000. This marked difference in antiherpetic activity between macrophages and fibroblasts was not observed with Foscarnet and PMEA. We suggest that this potent antiviral effect of acyclovir is mainly due to a proficient phosphorylation of the drug and/or a favourable dGTP/acyclovir triphosphate ratio in macrophage cells.     

Factors affecting cell viability during bisection of bovine embryos

The aim of this study was to investigate the effects of temperature, cytochalasin B, sucrose, cell number and developmental stage of embryos on cell loss and cell lysis during embryo splitting. Day-7 morulae and blastocysts were bisected using a metal blade. In Experiment 1, splitting of embryos in control medium (PBS + 10% fetal calf serum) was compared with splitting in the presence of 7.5 mul/ml cytochalasin B. In Experiment 2, the control medium was compared with medium supplemented with 200 mM sucrose. In Experiment 3, the control medium was compared with medium supplemented with sucrose and cytochalasin B. Cell viability was measured by staining nuclei of embryos with Hoechst 33258 and propidium iodide. Cells with nuclei exhibiting pink fluorescence were considered lysed, while blue fluorescence was considered an indication of viable cells. Cells disaggregated during splitting were classified as extruded cells. An effect of the developmental stage was observed in the pooled data from the control groups of the 3 experiments, with a higher proportion of viable cells in bisected morulae compared with bisected blastocysts (77.6 vs 70.0%; P = 0.003). However, as there was no effect of cell number (P = 0.85), the influence of the developmental stage can be contributed to morphological changes rather than to increase of cells associated with this change. In Experiment 1, the cytochalasin B-treated embryos contained a higher percentage of viable cells than the control embryos after removal of the developmental stage effect (P < 0.01). In Experiment 2, no effect on sucrose could be observed. In Experiment 3, the combined use of sucrose and cytochalasin B tended to increase the proportion of cells surviving bisection, but this difference was not significant. In Experiment 1, there was a correlation between viable cells and temperature during splitting (r = 0.42, P = 0.05; temperature range 8.1 degrees C to 15.6 degrees C). No correlation was found in any other group in any of the experiments, nor in the pooled data from the control groups in the 3 experiments.     

Factors affecting viable cell counts of freeze-driedCryptococcus terricolus cells

Freeze-drying ofCryptococcus terricolus cells in distilled water resulted in a survival of only 0.1% of the cells. The viability could be increased to 16% by the use of a dextran-sucrose-sodium glutamate solution as suspending medium.For freeze-dried material with both low and high survival rates, and for five as well as for ten days old cultures, malt extract solution was the superior reconstitution medium. Less, but still distinct, protective action was found with a synthetic glucose-urea-salt solution.The viable cell counts of cells freeze-dried in dextran-sucrose-sodium glutamate solution were independent of the medium used for plating. When distilled water was used as a medium for freeze-drying, two to four times higher counts were obtained with malt extract agar than with synthetic glucose-urea-salt agar.Of the twenty different media tried for freeze-drying, sucrose solution gave the best protection. The viability was greatly influenced by the concentration used, maximum values being obtained when more than 10% of sucrose was added. The survival rate increased with the age of the cells until the fifth day, but was independent of the concentration of cells in the suspension. Under optimum conditions a survival rate of more than 80% was reached.     

Expression of the leptin receptor in different types of vascular lesions

Clinical and experimental evidence suggests that the adipokine leptin may be important for the development of cardiovascular complications associated with obesity, possibly through interaction with its receptor on vascular cells. In the present study, we systematically analysed expression of the leptin receptor in normal and diseased vascular specimens using immunohistochemistry, immunofluorescence and quantitative real time-PCR. In particular, human atherosclerotic plaques as well as experimental vascular lesions induced in hypercholesterolemic mice and minipigs, respectively, were examined. Our results demonstrate the presence of the leptin receptor in normal vessel wall segments as well as neointimal or atherosclerotic lesions. In the latter, ObR expressing cells were predominantly localised on the luminal border and within the subintima, and coexpression of von Willebrand factor, VEGF receptor-2 or VE cadherin identified them as endothelial cells. Moreover, CD14-positive monocytes/macrophages were strongly positive for the leptin receptor. In contrast, only few ObR-expressing smooth muscle cells could be detected in human atherosclerotic plaques. The findings of the present study thus support a possible action of leptin on the cardiovascular system by demonstrating expression of the leptin receptor in different types of vascular lesions.     

Tolerance of different cell types for hypotonic action

The behavior of cells with different structure of cytoskeleton was studied in the hypotonic media: protoplasts, embryonal mouse fibroblasts and transformed mouse fibroblasts (L-line). Protoplasts were most sensitive to the hypotonic media. They began to disrupt in the hypotonic medium 4:1, and in the hypotonic medium 1:7 a complete lysis of cells was observed. Transformed mouse fibroblasts were disrupted in the medium diluted 1:15, while embryonal mouse fibroblasts were not disrupted in the medium diluted 1:31. Moreover, in the hypotonic conditions an essential difference was observed between the cells studied. Embryonal mouse fibroblasts are more tolerant to the hypotonic conditions than L-cells and protoplasts. It is suggested that the cytoskeleton may define the cell tolerance in hypotonic media.     

Factors affecting current production in microbial fuel cells using different industrial wastewaters

This study evaluated how different types of industrial wastewaters (bakery, brewery, paper and dairy) affect the performance of identical microbial fuel cells (MFCs); and the microbial composition and electrochemistry of MFC anodes. MFCs fed with paper wastewater produced the highest current density (125 ± 2 mA/m²) at least five times higher than dairy (25 ± 1 mA/m²), brewery and bakery wastewaters (10 ± 1 mA/m²). Such high current production was independent of substrate degradability. A comprehensive study was conducted to determine the factor driving current production when using the paper effluent. The microbial composition of anodic biofilms differed according to the type of wastewater used, and only MFC anodes fed with paper wastewater showed redox activity at −134 ± 5 mV vs NHE. Electrochemical analysis of this redox activity indicated that anodic bacteria produced a putative electron shuttling compound that increased the electron transfer rate through diffusion, and as a result the overall MFC performance.     

Factors affecting human cytomegalovirus gene expression in human monocyte cell lines

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.     

Factors affecting the viability and properties of microorganisms in different preservation methods

The review of literature devoted to the influence of the different methods of longterm preservation on the survival, physiological and biochemical properties of microorganisms: at low and ultralow temperatures, freeze-drying, drying, storage under the mineral oil etc. is given. The microorganisms viability depends on their nature, age and density of population, storage and recovery conditions of cells. Some features of the industrial microorganisms storage have been marked. The different hypotheses concerning the mechanism of preservation, injury and reactivation of microorganisms under the action of external factors during their storage are being discussed.