Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Neutral Red”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,567 records · Page 87Linked to original sources
PubMed2001

Quaternary ammoniums and other preservatives' contribution in oxidative stress and apoptosis on Chang conjunctival cells.

PURPOSE: To investigate some of the toxicity mechanisms of 10 preservatives currently used in ophthalmic solutions in vitro. METHODS: A continuous human conjunctival cell line was treated with different concentrations of various preservatives for 15 minutes and for 15 minutes followed by 24 hours of cell recovery: three benzalkonium chlorides (BACs) with different hydrocarbon chain length, benzododecinium bromide (BOB), cetrimide (Cet), phenylmercuric nitrate (PM), thimerosal (thi), methyl parahydroxybenzoate (MPHB), chlorobutanol (clb), and EDTA. An inhibition study was then conducted using a 1-hour vitamin E pretreatment followed by a 15-minute BAC treatment. Membrane integrity was assessed using a neutral red test and chromatin condensation with a Hoechst 33342 test. Reactive oxygen species were measured using dichlorofluorescein diacetate test for H2O2 production and hydroethidine test for O2.- production. These tests were performed using microplate cold light cytofluorometry. Cell size and DNA content were also analyzed using flow cytometry. Confocal microscopy was used to explore morphologic changes. RESULTS: A significant decrease of membrane integrity with chromatin condensation was observed with all the quaternary ammoniums tested at concentrations of 0.005% and higher. The effect was amplified after 24 hours of cell recovery. The other preservatives tested did not decrease membrane integrity. H2O2 production was observed with all the preservatives, whereas O2.- production was significantly higher with the quaternary ammoniums at 0.005% and 0.01%, compared with the other preservatives. Flow cytometry results confirmed the cytotoxicity observed with cold light cytofluorometry. CONCLUSIONS: The quaternary ammoniums tested (BAC, BOB, and Cet) were the most cytotoxic preservatives in the current model. An apoptotic mechanism appeared to be present at low concentrations of quaternary ammoniums, whereas a necrotic process appeared at higher concentrations. Superoxide anions may play an important role in tissue damage induced by preservatives in ocular surface disorders.

Apoptosis↗
PubMed2001

[Antioxidant effects of mast cell inhibitors in a human conjunctival cell line].

PURPOSE: The aim of this study was to evaluate in vitro antioxidant effects of two mast cells inhibitors. METHODS: Cytotoxicity tests were done on a continuous human conjunctival cell line using microplate cold light cytofluorimetry. Membrane integrity (neutral red test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluoresceine diacetate and hydroethidine tests) were evaluated on living cells treated with sodium cromoglycate and N-acetyl-aspartyl glutamic acid (NAAGA) preserved (benzalkonium chloride: BAC at 0.01%) and unpreserved after 60 minutes of treatment or 60 minutes and 24 hours of cell recovery. They were tested pure and at a 1/10 dilution. ROS production was also evaluated after a 60 minute pretreatment with antiallergic drugs and a 15-minute treatment with BAC, according to previous experiments performed on BAC showing its ROS production properties. RESULTS: No cytotoxicity was observed with the unpreserved formulations of antiallergic drugs. An apoptotic phenomenom was suggested with preserved drugs after a 1-hour treatment, whereas a necrotic mechanism appeared after a 24-hour cell recovery period. A ROS production decrease was observed with the two preserved and unpreserved drugs tested (p<0.001 compared to BAC) even if it was significantly higher with cromoglycate formulations. A ROS production decrease also was detected after a pretreatment with antiallergic drugs and treatment with BAC (p<0.001 compared to BAC alone). CONCLUSION: In vitro, no cytotoxicity was found with the two unpreserved mast cell inhibitors tested. An antioxidant effect also was observed with these two molecules; sodium cromoglycate appeared to be the best free radical scavenger.

Anti-Allergic Agents↗
PubMedDate not supplied

Chitosan-polyvinyl pyrrolidone hydrogel does not activate macrophages: potentials for transplantation applications.

We have shown previously that chitosan-polyvinyl pyrrolidone (PVP) hydrogels are blood compatible, islet compatible, and noncytotoxic to various cell types. Because of these potential applications of chitosan-PVP hydrogel, the present study was designed to investigate its effect on macrophage activation. Macrophages did not adhere to hydrogel in culture but maintained their viability and did not undergo apoptosis as confirmed by trypan blue staining and absence of DNA ladder. Hydrogel leach-out products did not exhibit cytotoxic effects on macrophage functionality at mitochondrial and lysosomal level as confirmed by tetrazolium reduction (MTT) and neutral red uptake (NRU) assay. On exposure to hydrogels, macrophages showed comparable expression of activation markers such as CDllb/CD18 (Mac-1), CD45, and CD14 to those cultured in the presence of PTFE, a known biocompatible control, indicating its nonactivating nature. Macrophage activation was also assessed by checking the level of messenger RNA of inflammatory cytokines such as IL-6 and TNF-alpha by reverse transcriptase polymerase chain reaction (RT-PCR), which did not show stastistically significant difference (p > 0.05) in the expression of these transcripts in both control and hydrogel-exposed macrophages. The nonimmunogenic nature of the hydrogel was further confirmed by the lack of induced proliferation of mouse splenic lymphocytes after exposure to hydrogel leach-outs. All these results point out that chitosan-PVP hydrogel did not activate macrophages and thus is immunocompatible. Our results indicate that this hydrogel could be a potential candidate for transplantation studies by virtue of its biocompatibility and imunocompatibility.

Animals↗
PubMed2001

Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture.

The antiviral effect of Australian tea tree oil (TTO) and eucalyptus oil (EUO) against herpes simplex virus was examined. Cytotoxicity of TTO and EUO was evaluated in a standard neutral red dye uptake assay. Toxicity of TTO and EUO was moderate for RC-37 cells and approached 50% (TC50) at concentrations of 0.006% and 0.03%, respectively. Antiviral activity of TTO and EUO against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of TTO for herpes simplex virus plaque formation was 0.0009% and 0.0008% and the IC50 of EUO was determined at 0.009% and 0.008% for HSV-1 and HSV-2, respectively. Australian tea tree oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of TTO plaque formation was reduced by 98.2% and 93.0% for HSV-1 and HSV-2, respectively. Noncytotoxic concentrations of EUO reduced virus titers by 57.9% for HSV-1 and 75.4% for HSV-2. Virus titers were reduced significantly with TTO, whereas EUO exhibited distinct but less antiviral activity. In order to determine the mode of antiviral action of both essential oils, either cells were pretreated before viral infection or viruses were incubated with TTO or EUO before infection, during adsorption or after penetration into the host cells. Plaque formation was clearly reduced, when herpes simplex virus was pretreated with the essential oils prior to adsorption. These results indicate that TTO and EUO affect the virus before or during adsorption, but not after penetration into the host cell. Thus TTO and EUO are capable to exert a direct antiviral effect on HSV. Although the active antiherpes components of Australian tea tree and eucalyptus oil are not yet known, their possible application as antiviral agents in recurrent herpes infection is promising.

Antiviral Agents↗
PubMed2001

Cytochrome P450 induction by nitrated polycyclic aromatic hydrocarbons, azaarenes, and binary mixtures in fish hepatoma cell line PLHC-1.

Nitrated polycyclic aromatic hydrocarbons (NPAHs) and N-heterocyclic aromatic hydrocarbons (azaarenes) are as ubiquitous in the environment as their parent PAH compounds, although occurring at lower concentrations. The toxicological importance of NPAHs and azaarenes is based on their mutagenic and carcinogenic potential. Azaarenes possess a higher solubility and mobility in the environment than PAHs. However, very little is known about the toxicity and cytochrome P450 (CYP)1A induction potencies of NPAHs and azaarenes in fish. Here we report on the cytotoxicities and relative CYP1A induction potencies of 12 NPAHs, 12 azaarenes, and 11 PAHs, determined as neutral red uptake and ethoxyresorufin-O-deethylase (EROD) activity, respectively, in fish hepatoma PLHC-1 cells. Additionally, CYP1A enzyme protein was determined by ELISA for two NPAHs, azaarenes, PAHs, and binary mixtures. Compared with the structurally analogous PAHs, 2-nitronaphthalene, 3-nitrofluoranthene, 2-aza- and 7-azafluoranthene, 1,6-dinitropyrene, benzo[a]acridine and benzo[h]quinoline revealed higher induction potencies, whereas the other compounds showed similar or less activity. The induction potency was highly dependent on the compounds structural properties, reflected by significant correlations between the half-maximal EROD induction (-log EC50) and the molecular descriptors lipophilicity (log Kow) and maximal molecular length (Lmax). Binary mixtures of 6-nitrochrysene + benzo[a]anthracene, 6-nitrochrysene + benzo[a]acridine, and benzo[a]acridine + benzo[a]anthracene showed an additive interaction. The CYP1A induction potencies of NPAHs and azaarenes, demonstrated here for the first time in fish hepatoma cells, suggest that their contribution to the overall CYP1A induction potencies in PAH-contaminated environmental samples have to be taken into account.

Animals↗
PubMedDate not supplied

Aurintricarboxylic acid decreases proliferative potential of SKOV3 and MCF7 human carcinoma cells.

The effect of aurintricarboxylic acid (ATA) on cell growth and proliferative capacity was studied in human ovarian SKOV3 and breast MCF7 carcinoma cells. ATA moderately inhibited cell growth measured by a Neutral red assay after a 24-hour incubation of the cells in the presence of ATA. The ATA-treated cells displayed a markedly decreased capacity to proliferate, as was evident from a colony formation assay. The initial and delayed anti-proliferative effects of ATA were dose-dependent. Together, the results indicated that ATA offers the potential of being recognized as an anti-tumor drug, at least in certain types of cancers.

Antineoplastic Agents↗
PubMed2001

Mitochondrial activity and glutathione injury in apoptosis induced by unpreserved and preserved beta-blockers on Chang conjunctival cells.

PURPOSE: Quaternary ammonium ions have been demonstrated to induce apoptosis correlated with superoxide anion production in vitro. The purpose of this study was to further investigate the mechanisms of benzalkonium chloride (BAC), unpreserved and preserved beta-blocker eye-drops-induced programmed cell death, with special attention to the roles of mitochondrial transmembrane potential and intracellular reduced glutathione. METHODS: Chang conjunctival cells were incubated with different concentrations of unpreserved or preserved timolol (0.1%, 0.25%, and 0.4%), or carteolol (1% and 2%), or BAC (0.0001% to 0.01%) for 15 minutes, or for 15 minutes with a 24-hour recovery period in normal medium. Cellular viability (neutral red test), mitochondrial activity (rhodamine 123 test), intracellular reduced glutathione (monochlorobimane test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests) were evaluated using microplate cold-light cytofluorometry. RESULTS: A significant, concentration-dependent decrease in cellular viability was found with preserved beta-blockers and with BAC alone, whereas unpreserved preparations did not show any toxicity. Only preserved beta-blockers induced chromatin condensation associated with an alteration of mitochondrial activity and a decrease of glutathione, suggesting an apoptotic phenomenon. BAC increased glutathione after 15 minutes, whereas a decrease was observed after a recovery period. ROS production was found with preserved formulations at significantly higher levels than those observed with unpreserved drugs. CONCLUSIONS: This in vitro study demonstrates that oxidative stress, evidenced by enhanced ROS production and mitochondrial injury rather than by cellular glutathione depletion, is a mechanism involved in apoptosis induced by preservative-containing eye-drops.

Adrenergic beta-Antagonists↗
PubMed2001

[Effects of nitric oxide and prostaglandin on gastric mucosal perfusion in rats with portal hypertensive gastropathy].

OBJECTIVE: To observe the effects of nitric oxide (NO) and prostaglandin on gastric mucosal perfusion in rats with portal hypertensive gastropathy (PHG). METHODS: Two weeks after partial portal vein ligation, gastric mucosal blood flow (GMBF) was measured with the neutral red clearance method and the changes of portal venous pressure (PVP) were observed. RESULTS: GMBF and PVP were significantly higher in rats with PHG than in sham operated rats (P<0.01). Low dose inhibitor of NO synthesis N(-nitro-L-arginine methyl ester (L-NAME 1mg/kg, 4mg/kg) caused a significant and dose dependent reduction in GMBF in PHG rats, but had no effect on sham operated rats. High dose L-NAME (12 mg/kg) significantly decreased GMBF in both PHG and sham operated rats. The inhibition of prostaglandin synthesis with indomethacin significantly decreased GMBF in rats with PHG, but had no effect in sham operated rats. In sham operated rats pretreated with indomethacin, GMBF was not modified after and before injection of low dose L-NAME (4mg/kg); likewise, high dose of L-NAME (12mg/kg) did not alter the significant reduction in GMBF as compared with pretreatment without indomethacin. In PHG rats the significant dose dependent reduction in GMBF induced by L-NAME (4mg/kg, 12mg/kg) was not significantly different between pretreatment with and without indomethacin. CONCLUSIONS: NO and prostaglandins play an important role in the regulation of GMBF in rats with PHG, but no synergistic interactions between them.

Animals↗
PubMed2001

Role of calcium, glutamate neurotransmission, and nitric oxide in spreading acidification and depression in the cerebellar cortex.

This study investigated the mechanisms underlying the recently reported fast spreading acidification and transient depression in the cerebellar cortex in vivo. Spreading acidification was evoked by surface stimulation in the rat and mouse cerebellar cortex stained with the pH-sensitive dye neutral red and monitored using epifluorescent imaging. The probability of evoking spreading acidification was dependent on stimulation parameters; greater frequency and/or greater amplitude were more effective. Although activation of the parallel fibers defined the geometry of the spread, their activation alone was not sufficient, because blocking synaptic transmission with low Ca(2+) prevented spreading acidification. Increased postsynaptic excitability was also a major factor. Application of either AMPA or metabotropic glutamate receptor antagonists reduced the likelihood of evoking spreading acidification, but stronger stimulation intensities were still effective. Conversely, superfusion with GABA receptor antagonists decreased the threshold for evoking spreading acidification. Blocking nitric oxide synthase (NOS) increased the threshold for spreading acidification, and nitric oxide donors lowered the threshold. However, spreading acidification could be evoked in neuronal NOS-deficient mice (B6;129S-Nos1(tm1plh)). The depression in cortical excitability that accompanies spreading acidification occurred in the presence of AMPA and metabotropic glutamate receptor antagonists and NOS inhibitors. These findings suggest that spreading acidification is dependent on extracellular Ca(2+) and glutamate neurotransmission with a contribution from both AMPA and metabotropic glutamate receptors and is modulated by nitric oxide. Therefore, spreading acidification involves both presynaptic and postsynaptic mechanisms. We hypothesize that a regenerative process, i.e., a nonpassive process, is operative that uses the cortical architecture to account for the high speed of propagation.

Acids↗
PubMed1999

[Effect of cefodizime on cellular immunity in patients with lung cancer].

OBJECTIVE: To study the enhancing effect of cefodizime on cellular immunity of patients with lung cancer. METHODS: The parameters of cellular immunity in blood and pulmonary alveoli were investigated in 45 patients with lung cancer complicated with lower respiratory tract infection (LRTI) and 10 non-cancer patients with LRTI. Nitroblue tetrazolium (NBT) reaction of polymorphonuclear neutrophils (PMN), the percentage of natural killer (NK) cells, and that of CD4+ cells, ratio of CD4+/CD8+ in peripheral blood, OD value of alveolar macrophages phagocyting neutral red, and IL-1 beta (ng/L) in broncho-alveolar lavage flind (BALF) were determined. RESULTS: The parameters examined in cancer patients with LRTI were significantly improved after cefodizime treatment, approaching to those seen in non-cancer patients with LRTI. The therapeutic results were better in patients treated with cifodizime than in patients treated with ceftriaxonum. CONCLUSION: Cefodizime is a better choice of antibiotic for the treatment of lung cancer complicated with lower respiratory tract infection. Its enhancing effect on systemic and local immune functions plays a role.

Adult↗
PubMed2001

In vitro study on the morphology of human blood dendritic cells and LPAK cells inducing apoptosis of the hepatoma cell line.

OBJECTIVE: To observe in vitro effects and morphological changes of human peripheral blood dendritic cells (DCs) on the ability of lymphokine and phytohaemagglutininum (PHA) activated killer (LPAK) cells to induce apoptosis of the human hepatoma cell line (BEL-7402, B). METHODS: Experimental groups were divided into LD group (DCs + L + B), L group (L + B), D group (DCs + B) and B group. The methods of neutral red uptake, ordinary light microscopy, electron microscopy, TDT mediated X-dUTP nick end labeling (TUNEL) were used. RESULTS: The difference between the D group and the B group was not distinct (P > 0.05). The difference between the LD group and the L group was distinct, with DCs + LPAK > LPAK (P < 0.01) in cytotoxicity. Apoptotic cells were TUNEL positive in light microscopy, and apoptotic nuclei were stained yellow brown and dark brown, with size and shape varying from cell to cell. Ultrastructural change in apoptotic tumor cells comprised of compaction and condensation of nuclear chromatin, and condensation of cytoplasm and apoptotic bodies. At the same time, LPAK cells manifested the characteristics of autophagic apoptosis, and there were some autophagic bodies in it. CONCLUSIONS: The combination of human blood DCs and LPAK cells could induce apoptosis of BEL-7402 cells effectively, with some LPAK cells manifesting the characteristics of autophagic apoptosis.

Apoptosis↗
PubMed2001

[Subcellular distribution of daunorubicin in the P-glycoprotein-mediated multidrug-resistant cell line K562/ADR].

OBJECTIVE: To examine subcellular distribution of daunorubicin (DNR) in P-glycoprotein-mediated multidrug-resistant cell line K562/ADR and its relation to multidrug resistance. METHODS: The subcellular distribution of DNR in K562/ADR was studied by confocal scanning laser microscopy (CSLM), fluorometry, RT-PCR. Rhodamine 123, NBD-ceramide and neutral red as fluorescent probes to stain the mitochondria, Golgi apparatus and lysosomes respectively were used to identify the subcellular compartments wherein DNR was sequestered. Effect of verapamil, chloroquine and brefeldin A on DNR distribution and accumulation was examined. RESULTS: Compared with the drug-sensitive cell K562/S in which DNR fluorescence diffusely appeared in the nucleus and cytoplasm, DNR in K562/ADR cells was distributed to the perinuclear region and peripheral cytoplasm, It was scenty in the nucleus and other cytoplasmic regions, as suggested by the distribution of Rhodamine123. Only verapamil, but not chloroquine and brefeldin A, could markedly restore diffuse cytoplasmic and nuclear fluorescence distribution in the resistant cell line. CONCLUSION: Altered subcellular distribution of DNR in drug resistant cell line may participate in the generation of multidrug resistance in which P-glycoprotein plays an important role.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

In vitro biocompatibility evaluation of naturally derived and synthetic biomaterials using normal human bladder smooth muscle cells.

PURPOSE: Tissue engineering of the urinary tract often requires the use of various biomaterials. Adequate biomaterial biocompatibility is necessary for successful urinary reconstruction. In this study using a primary normal human bladder smooth muscle cell culture system we evaluated the in vitro biocompatibility of a number of naturally derived biomaterials, including bladder submucosa, small intestinal submucosa, collagen and alginate, and polymeric biomaterials, including polyglycolic acid, poly(L-lactic acid) and poly(lactic-co-glycolic acid, which have been used for urinary reconstruction experimentally or clinically. MATERIALS AND METHODS: To determine the cytotoxic and bioactive effects of bladder submucosa, small intestinal submucosa, collagen, alginate, polyglycolic acid, poly(L-lactic acid) and poly(lactic-co-glycolic acid) we measured cell viability, metabolic activity, apoptotic properties and DNA synthesis activity with 4 types of assays, namely Neutral Red (Sigma Chemical Co., St. Louis, Missouri), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma Chemical Co.), apoptotic activity and tritiated thymidine incorporation (Dupont NEN, Boston, Massachusetts) assays. Normal human bladder smooth muscle cells were cultured with the extracts of the biomaterials or cultured in direct contact with the biomaterials. RESULTS: All naturally derived and synthetic biomaterials tested in this study except alginate exhibited nontoxic and bioactive effects on human bladder smooth muscle cells (HBSMCs) in vitro, as indicated by the 4 types of biocompatibility assays using the extract and direct contact methods. Cell viability, apoptotic properties, metabolic activity and DNA synthesis activity of HBSMCs cultured with the extracts of the biomaterials or cultured in direct contact with the biomaterials were not significantly different from those of negative controls (fresh medium with no extracts or tissue culture plates without biomaterials). CONCLUSIONS: All naturally derived and synthetic biomaterials tested in this study except alginate exhibited nontoxic and bioactive effects on HBSMCs in vitro. This normal primary human bladder smooth muscle cell culture model is suitable for in vitro biocompatibility assessment. It provides information on cell-biomaterial interactions and on the ability of biomaterials to support bioactive cell functions.

Biocompatible Materials↗
PubMed2001

[The inhibitory effect of decomposed Chinese traditional medicine Chaihu on Coxsackie B virus(CVB3m) replication and its influence on cell activity].

OBJECTIVE: To study the anti-Coxsackie B virus (CVB3m) action of Chaihu(XCT) and its decomposed herb soups No.1 and No.2 in vitro, and also their protective effect on cells. METHODS: The anti CVB3m and cell protection effects of XCT and its decomposed herb soups No.1 and No.2 were observed by the methods of micro-cell culture and neutral red ingestion, taking cytopathic effect and cell activity as judgments of medicine toxicity and virus replication. RESULTS: The non-toxic concentrations of XCTand its decomposed herb soups No.1 and No.2 had no apparent influence on HeLa cell activity, on the contrary, in certain range of concentrations, they could promote cell growth and cell activity. In therapeutic cell group, XCT and its decomposed herb soups No.1 and No.2 all had apparent inhibitory effect on CVB3m replication, especially the decomposed No.1 showing an inhibitory rate of 107.6%. Under the same decomposed No.1 concentration(1.5 mg/ml), the viral inhibitory rate of the preventive therapeutic cell group was much higher than that of the therapeutic cell group, reaching as high as 128.1%. In virus adsorbed cell group, the CVB3m was also obviously inhibited by the XCT and decomposed No.1 and No.2. CONCLUSIONS: By comparing the effects on cell protection and virus replication of XCT and its decomposed herb soups No.1 and No.2, it identifies that XCT can protect cells against virus infection and directly kill the CVB3m, this Chinese herb medicine may be applied clinically for preventing and curing of viral myocarditis.

Antiviral Agents↗
PubMed2002

Anatomical and functional study of localization of originating neurons of the parasympathetic nerve to gallbladder in rabbit brain stem.

The present study was to investigate the localization of preganglionic parasympathetic neurons of gallbladder in brain stem by anatomical and functional approaches. Male or female rabbits (n = 11) were anesthetized with sodium pentobarbital (30 mg/kg, i.v.). Cholera toxin B conjugated to horseradish peroxidase (CB-HRP) was injected into the gallbladder wall. Four days later, animals were re-anesthetized and perfused transcardially with paraformaldehyde solution in a 0.1 M phosphate buffer. The rabbit brain was then frozenly sectioned. The sections were processed for HRP label and stained with neutral red. Another group of rabbits (n = 54) were anesthetized by urethane (1 g/kg) after fasting for 18-24 hours, Gallbladder pressure (GP) was measured by inserting a frog bladder filled with normal saline into the gallbladder. Myoelectrical activity of the sphincter of Oddi (SO) was induced by a pair of copper electrodes. A glass tube (30 microm tip diameter) connected with a microsyringe was directed to the dorsal vagal complex (DVC) for microinjection. Majority of retrogradely labeled cells was found bilaterally in dorsal motor nucleus of the vagus nerve (DMV) throughout the length, except the rostral and caudal part. These cells were distributed in subnuclei parvicellularis or mediocellularis of DMV. Some labeled perikarya located in the medial subnucleus of the solitary tract (mNTS). Thyrotropin-releasing hormone (TRH, 1.3 mmol/L, 0.2 microl) microinjected into the rostral portion of the DVC (including DMV and NTS) enhanced the motility of gallbladder and SO. Microinjection of TRH at the middle part of DVC seldom induces excitatory effects on the gallbladder or SO. TRH microinjected into the caudal portion of the DVC elicited weaker response of gallbladder and SO than rostral portion. Our results indicated that DMV is one of the most important original nuclei of gallbladder's vagus nerves and mNTS may be also involved in the control of gallbladder's parasympathetic activity. Neurons that innervate the gallbladder distribute at most part of DVC, and are relatively dense at rostral and caudal position of DMV.

Animals↗
PubMed2002

Cytoprotective effects of hyaluronic acid and Carbomer 934P in ocular surface epithelial cells.

PURPOSE: To investigate in vitro the cell toxicity and antioxidant effects of two major tear substitutes, hyaluronic acid and a widely used carbomer, with and without preservative. METHODS: Chang conjunctival cells were treated with different concentrations of unpreserved or preserved carbomer 934P (0.03% and 0.3%), unpreserved or preserved hyaluronic acid (0.018% and 0.18%), and benzalkonium chloride (BAC 0.0005% and 0.005%) for 15 minutes or for 15 minutes with 24 hours of cell recovery, according to previously validated methods. Microplate cold light cytofluorometry was performed to evaluate cell viability (neutral red test), chromatin condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests). Confocal microscopy was used to explore morphologic changes. RESULTS: No alterations were found with unpreserved and preserved hyaluronic acid at all concentrations and times tested. A decrease in cell viability with chromatin condensation appeared with 0.3% preserved carbomer 934P at the two times tested. This cytotoxicity, however, was significantly less than that observed with BAC alone, although the same concentrations of preservative were used. Unpreserved carbomer 934P induced no modification of cell viability after 15 minutes but a significant decrease in chromatin condensation, reversible after 24 hours of cell recovery, when a delayed decrease in cell viability was observed. Production of reactive oxygen species (ROS) decreased with the four formulations of tear substitutes tested at their usual concentrations, whereas a significant production of ROS occurred with BAC. CONCLUSIONS: These two ophthalmic hydrogels have no cytotoxicity but possess antioxidant properties and tend to reduce the toxic effects of preservatives. These results may allow use of hydrogels, not only in dry eye but also in ocular surface disorders involving oxidative stress and in ophthalmic drug therapy to improve ocular tolerance.

Acrylic Resins↗
PubMed2002

Distribution of furamidine analogues in tumor cells: targeting of the nucleus or mitochondria depending on the amidine substitution.

Diphenylfuran diamidines represent an important class of DNA minor groove binders of high therapeutic interest as antiparasitic or antitumor agents depending on the compounds structures. To exert their cytotoxic action, the compounds must first get into the cell and reach the nuclear compartment where the main target, DNA, is located. The forces that drive the drugs into cell nuclei, as well as the influence of the molecular structures on the cell distribution, are not known. To address these issues, we took advantage of the fluorescence of the molecules to analyze their intracellular distribution profiles in tumor cells of different origins (B16 melanoma, MCF7 mammary adenocarcinoma, A549 lung carcinoma, HT29 colon carcinoma, LNCaP, and PC3 prostatic carcinoma) by epifluorescence and confocal microscopy. A homogeneous series of synthetic bis-substituted alkyl or phenyl amidine and reverse amidine derivatives of furamidine was used to dissect the molecular mechanisms that control the distribution of the drugs into the cytoplasm or the nucleus of the cells. The amidine (DB75) and the various N-alkyl derivatives were found to accumulate selectively in the cell nuclei. This is also the case for a guanidine derivative but not for the phenyl-substituted compound DB569, which essentially localizes in cytoplasmic granules. Similar cytoplasmic patterns were observed with a reverse amidine analogue and a pyridine-substituted compound indicating that the presence of aromatic rings on the terminal side chain is the limiting factor that restricts the uptake of the compounds in the nuclear compartment. The use of different organelle-selective fluorescent probes, such as JC-1 and chloromethyl-X-rosamine, both specific to mitochondria and neutral red considered as a lysosome-selective probe, suggests that DB569 preferentially accumulates in mitochondria. Competition experiments with the antitumor drug daunomycin reveal that the diphenylfurans are attracted into the nuclei by the DNA. The DNA minor groove-drug interactions provide the driving force that permits massive accumulation of the fluorescent molecules in the nuclei. The DNA binding properties of the diphenylfuran derivatives were investigated by DNase I footprinting and surface plasmon resonance biosensor experiments to measure sequence selectivity and binding affinities, respectively. Furamidine and its phenyl-substituted analogue that accumulate in the cell nuclei and mitochondria, respectively, share a common selectivity for AT sites and bind equally tightly to these sites. Therefore, it is possible to modulate the intracellular distribution of the furamidine derivatives without affecting their DNA binding and sequence recognition properties. The introduction of aromatic substituents on diphenylfuran diamidines represents a novel strategy to control the intracellular compartmentalization of these DNA binding agents and directs them to mitochondria. This drug design strategy may prove useful to trigger drug-induced apoptosis.

Adenocarcinoma↗
PubMed2002

In vitro studies of activation of phagocytic cells by bioactive peptides.

The effect of formyl chemotactic peptide (fCTP, fMet-Leu-Phe), beta-amyloid peptides (beta-AP, 1-42, 1-16 and 25-35), and bradykinin (BK) on functional activity of phagocytic cells has been investigated. Wheat germ agglutinin (WGA) was also used as a model membrane binding agent of polypeptide nature. Murine monocyte-macrophage cell line J774.2 and normal human blood polymorphonuclear (PMN) cells were used as target phagocytic cells. Their activity was quantitatively estimated by measuring phagocytosis of killed yeast cells. Beta-AP (1-41) maximally stimulated phagocytosis at 0.1 microg/ml, BK--at 1.0 microg/ml, and fCTP--at 2.0 microg/ml. Beta-AP (1-16) and beta-AP (25-35) were inactive in used test-systems. Phagocytosis-inducing activity of beta-AP (1-42) and BK reached maximal levels in 2 h and decreased after 4-6 h of incubation. Phagocytosis numbers were compared with the indicators of phagocytic cell activation, such as absorption of neutral red dye, glucose utilization, production of super-oxide anion (NBT-test) and nitrite accumulation (indicator of NO production). NBT-test, which may be related to the killing ability of phagocytic cells towards the ingested objects, was positive only in stimulated PMN leukocytes, while the nitrite accumulation was detected only in stimulated macrophages. Nitrite accumulation in macrophages was markedly induced by lipopolysaccharide and to a lower extent by 0.5 microg/ml beta-AP (1-42). In high dose (5.0 microg/ml) beta-AP suppressed nitrite accumulation in macrophages stimulated by lipopolysaccharide. Other studied peptides were inactive in inducing nitrite accumulation. Transforming growth factor type beta suppressed phagocytic activity of PMN cells activated by beta-AP or WGA. The anti-inflammatory drugs (indomethacin and L-lysine aescinate) inhibited beta-AP (1-42)-induced phagocytosis. The interrelations between the regulatory pathways of BK, beta-AP and fCTP are discussed.

Amyloid beta-Peptides↗