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At least 1,423 records · Page 79Linked to original sources
PubMed1983

The role of 5-hydroxytryptamine as a transmitter between identified leech neurones in culture.

The synthesis, storage, release and synaptic actions of 5-hydroxytryptamine (5-HT or serotonin) were studied in order to characterize the synaptic connexion that develops between pairs of identified neurones dissected from the central nervous system of the leech and maintained in culture. Experiments were made with Retzius cells (which are known to contain 5-HT in vivo) and pressure sensory neurones on which they form chemical synapses in culture. Individual, isolated Retzius cells in culture synthesized [3H]5-HT from either [3H]tryptophan or [3H]5-hydroxytryptophan [( 3H]5-HTP). These cells did not synthesize other putative neurotransmitters, such as acetylcholine, dopamine, octopamine, noradrenaline or gamma-aminobutyric acid from their respective precursors. The monoaminergic character of Retzius cells was also demonstrated by staining with Neutral Red and by histofluorescence. Individual, isolated Retzius cells that had synthesized and accumulated [3H]5-HT released this compound when depolarized. Transmitter release was calcium-dependent and was blocked by magnesium. When incubated with [3H]5-HT and washed, Retzius cells in culture accumulated approximately 100 times more labelled 5-HT than did non-serotonergic cells, and 10 times more than Retzius cell somata acutely isolated from the animal and incubated in vitro. Chlorimipramine, a blocker of 5-HT uptake, decreased the amount of [3H]5-HT accumulated by Retzius cells and also caused a reversible increase in the amplitude of the synaptic response in the pressure sensory cell elicited by stimulation of the Retzius cell. Pressure sensory neurones in culture and in vivo responded to 5-HT focally applied by pressure ejection from a micropipette. Small pulses elicited a small, slow hyperpolarization. This response was due, at least in part, to an increase in chloride conductance and desensitized rapidly. With larger pulses, a larger, faster non-desensitizing depolarization was elicited. Together, these results provide evidence that 5-HT released from Retzius cells could be responsible for the chemical synaptic potentials seen in pressure sensory neurones in culture.

5-Hydroxytryptophan↗
PubMed2003

In vitro discrimination of fluoroquinolones toxicity on tendon cells: involvement of oxidative stress.

Tendinopathy are classic side effects observed with fluoroquinolones antibiotics. A previously validated model based on a spontaneously immortalized rabbit tendon cell line (Teno cell line) was used to evaluate cellular responses to the fluoroquinolones pefloxacin (PEF), ofloxacin (OFX), levofloxacin (LVX), and ciprofloxacin (CIP), in various concentrations. Cell viability, redox status changes, reduced glutathione content, and reactive oxygen species production were assessed using neutral red, Alamar blue, monobromobimane and 2,7-dichlorofluorescindiacetate fluorescent probes, respectively. Living adherent tenocytes were analyzed using a cold light cytofluorometer adapted to 96-well microplates. All fluoroquinolones showed moderate cytotoxicity after 24 h and more severe, significant toxicity after 72 h on tendon cells. Moreover, two groups of fluoroquinolones may be differentiated: intrinsic toxicity for tendon cells was high with ciprofloxacin and pefloxacin [redox status decrease was 80 and 62% (*p < 0.05) for PEF and CIP at 1 mM for 72 h, respectively], but moderate with ofloxacin and levofloxacin LVX [redox status decrease was 30 and 22% (*p < 0.05) for OFX and LVX at 1 mM during 72 h, respectively]. Our model supports a role for early oxidative stress in the development of fluoroquinolone-induced tendinopathy. Moreover, our study indicates that intrinsic toxicity to tendon cells varies across fluoroquinolones. The Teno cell line may be a useful model for detecting and evaluating tendon toxicity of new fluoroquinolones and other drugs associated with tendinopathy.

Animals↗
PubMed2002

Beta-lyase-dependent attenuation of cisplatin-mediated toxicity by selenocysteine Se-conjugates in renal tubular cell lines.

Cisplatin [cis-diamminedichloroplatinum(II)] is a widely used antitumor drug with dose-limiting nephrotoxic side effects due to selective toxicity to the proximal tubule. In the present study, the chemoprotective potential of three selenocysteine Se-conjugates, Se-methyl-L-selenocysteine, Se-(2-methoxyphenyl)-L-selenocysteine, and Se-(2-chlorobenzyl)-L-selenocysteine, belonging to three structural classes, against the nephrotoxic effects of cisplatin was investigated. Selenocysteine Se-conjugates have previously been proposed as kidney-selective prodrugs of pharmacologically active selenols because of their active uptake and bioactivation by cysteine conjugate beta-lyases in the kidney. To elucidate whether chemoprotection is beta-lyase-dependent wild-type LLC-PK(1) cells, possessing a very low beta-lyase activity, and LLC-PK(1) cells stably transfected with full-length cDNA coding for rat kidney cysteine conjugate beta-lyase/glutamine transaminase K (R1J) were used. The results indicate that all three selenocysteine Se-conjugates were able to attenuate the cisplatin-induced loss of viability in R1J cells but not in the parental LLC-PK(1) cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and neutral red uptake. In addition, cisplatin-induced reactive oxygen species (ROS) production was determined using 2',7'-dichlorodihydrofluorescein diacetate. The selenocysteine Se-conjugates were able to decrease ROS levels after cisplatin exposure in both cell types. However, this ROS-protective effect was more profound in R1J cells. Se-Methyl-L-selenocysteine provided the strongest protection. The protective activity against cisplatin-induced cytotoxicity and ROS generation was blocked by aminooxyacetic acid, a selective inhibitor of pyridoxal 5'-phosphate-dependent cysteine conjugate beta-lyases, further supporting the role of beta-lyase in the observed chemoprotection. The precise molecular mechanism by which selenols, generated by beta-lyase, provide protection against cisplatin-induced cytotoxicity, however, remains to be established.

Animals↗
PubMed1982

Antiviral activity and mechanism of action of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860).

A nitrobenzene derivative, MDL-860, was found to inhibit plaque formation, cytopathic effect, or both in 11 of 12 picornaviruses at concentrations which did not affect cell growth. The compound did not directly inactivate the virus. MDL-860 inhibited actinomycin D-resistant [3H]uridine uptake in cells infected with coxsackievirus A21 or rhinovirus 1-A, whereas incorporation into uninfected cells was not inhibited. With three picornaviruses (echovirus type 12, poliovirus type 2, and rhinovirus type 1-A) made photosensitive with neutral red, MDL-860 did not appear to cause a significant reduction in their loss of photosensitivity (uncoating) during the first 3 h of infection. MDL-860 appears to inhibit some early event in virus replication, after uncoating, which is required for synthesis of a majority of viral RNA.

Animals↗
PubMed1991

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.

Animals↗
PubMed1970

Dengue virus plaque development in simian cell systems. I. Factors influencing virus adsorption and variables in the agar overlay medium.

Chemical and physical variables influencing the plaquing of all dengue serotypes in two simian cell systems were studied. Calf serum in the nutrient overlay may be replaced by mouse ascitic fluid or bovine plasma albumin when employing the rhesus monkey kidney LLC-MK(2) cell system for plaquing all dengue serotypes. Doubling the serum concentration in the overlay had little effect in modifying dengue types 1, 2, 3, and 4 plaque titers. Newborn agamma, 4-week-old and 8-week-old calf serum gave comparable titers with all dengue virus serotypes. Dengue virus titers, plaque size, and development time were unaffected by sodium bicarbonate concentrations ranging from 1.1 to 4.4 mg/ml of overlay. A twofold increase (0.00332 g%) in the amount of either autoclaved or filtered-sterilized neutral red reduced the dengue 2 virus titer as much as 2.2 logs. An increased Mg(++) and decreased Ca(++) concentration in the overlay medium increased the efficiency of the plaquing system.

Adsorption↗
PubMed1970

Agar overlay plaquing technique for Pseudomonas aeruginosa in HeLa monolayers.

The solid agar overlay procedure used for viral plaquing was adapted to the study of Pseudomonas aeruginosa plaques in HeLa monolayers. After adsorption of the organism to the HeLa monolayers, an overlay consisting of 10% newborn calf serum (NBCS), 90% Eagles basal medium (EBM), and 1.5% agar containing neutral red was added. Plaques developed after 24 hr of incubation at 37 C which were indistinguishable from viral lesions. Optimal conditions for plaques included: serum in the adsorption fluid as well as the overlay medium; 1-hr adsorption time; healthy HeLa monolayers; and the presence of EBM. The formation of plaques instead of colonies in agar was due to the inhibitory effect of NBCS or EBM. By omitting both of these components from the overlay, colonies appeared in place of plaques. Adult bovine serum acted in a similar bacterial colony-suppressing fashion, but fetal or agamma calf serum did not. Furthermore, NBCS or adult bovine serum when incorporated in the overlay medium instead of fetal or agamma calf serum displayed a plaque-suppressing effect by reducing the number and size of plaques formed. The findings lend further evidence to support the hypothesis that P. aeruginosa may produce plaques from a protected intracellular site within HeLa cells.

Animals↗
PubMed1973

A rapid, presumptive procedure for the detection of Salmonella in foods and food ingredients.

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory.

Bacteriological Techniques↗
PubMed1974

Clinical evaluation of enteric media in the primary isolation of Salmonella and Shigella.

Over a 1-year period, media for the isolation of enteric pathogens were compared on 455 stool specimens. Fifty-three pathogens were isolated, of which 56% were Shigella sonnei and 13% were Sh. flexneri. Of these isolates, 90% were found on xylose-lysine-desoxycholate agar, 87% on Hekton enteric agar, and 80% on MacConkey without crystal violet with 2% agar and 0.007% neutral red, but only 28% were recovered on Salmonella-Shigella agar. Less than one-half of the shigellae were recovered after Selenite-F enrichment. On the other hand, enrichment was the most helpful method for isolating salmonellae. Studies on cultures from which mixed isolates were obtained indicated that numbers and chance distribution have an effect on the results obtained. The performance of Salmonella-Shigella agar in the isolation of enteric pathogens was inferior, and the effort involved to obtain those isolates was greater than for Hekton enteric and xylose-lysine-desoxycholate agars.

Agar↗
PubMed1976

Assay of bovine interferons in cultures of the porcine cell line IB-RS-2.

An assay for bovine interferons has been developed using the porcine cell line IB-RS-2 and a bovine enterovirus, CBV-D, as challenge virus. The method is based on estimation of cytopathic effect measured by uptake of neutral red. Teh assay is simple, sensitive, and reproducible. A comparative test of different viruses in IB-RS-2 cells and secondary calf kidney cells revealed that the sensitivity of a virus to interferon can vary up to 1,000-fold in the two cell systems. Vesicular stomatitis virus was found to be rather insensitive to interferon in IB-RS-2 cells.

Animals↗
PubMed1990

Helicobacter pylori urease activity is toxic to human gastric epithelial cells.

A human gastric adenocarcinoma cell line was used to evaluate the contribution of urease from Helicobacter (formerly Campylobacter) pylori to its cytotoxicity. Gastric cells cultured in medium supplemented with 20 mM urea were exposed to 5 x 10(6) CFU of H. pylori per ml with or without the addition of a urease inhibitor, acetohydroxamic acid. Viabilities of cells exposed to H. pylori for 2, 24, and 48 h, assessed by incorporation of neutral red dye, were 60, 27, and 16%, respectively; however, the viabilities of cells exposed to both H. pylori and acetohydroxamic acid were 92, 46, and 20% after 2, 24, and 48 h, respectively, (P less than 0.001). Therefore, the urease activity of H. pylori may play an important role in its pathogenicity, and inhibition of this enzyme activity may have therapeutic potential.

Ammonia↗
PubMed1996

Influence of Actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells.

The effect of Actinobacillus pleuropneumoniae and its metabolites on the viability of porcine alveolar epithelial cells was studied by using a neutral-red uptake test. Alveolar epithelial cells were obtained from 5-week-old colostrum-deprived pigs. The purity of these cells as assessed by the modified Papanicolaou stain was 90 to 95%. Incubation of these cells with 10(6) CFU of a biotype 1 serotype 1 strain resulted in death of the alveolar epithelial cells within 1.5 h. A cytotoxic effect was also seen when alveolar epithelial cells were incubated with sterile culture supernatants of biotype 1 serotype 1, biotype 1 serotype 10, and biotype 2 serotype 2 strains or with ApxI, ApxII, or ApxIII produced by recombinant Escherichia coli. Incubation of alveolar epithelial cells with a knockout mutant of the biotype 1 serotype 1 parent strain which is unable to secrete Apx toxins or with its supernatant did not result in death of these cells. These results indicate that cytotoxicity is at least in part due to production of Apx toxins.

Actinobacillus pleuropneumoniae↗
PubMed1997

Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells.

Cytolethal distending toxin (CDT) is a newly described toxin produced by a number of enteropathogens, including Campylobacter jejuni, various Escherichia coli strains, and a few Shigella species. CDT induces distension and eventual death of a number of transformed cell lines. Here, we extend previous studies by demonstrating that morphological changes in CDT-treated Chinese hamster ovary cells are coincident with changes in cytoskeletal structure and an inhibition of cell proliferation. CDT-treated cells underwent a progressive accumulation of F-actin assemblies which microscopically resembled actin stress fibers. Accumulation of the stress fiber-like structures in CDT-treated cells was accompanied by an apparent blockage of cell division. Multinucleation was detected in some cells but did not constitute a significant feature of CDT action. Although toxin-treated cells failed to divide, cell viability remained high for the first 4 days following toxin treatment, as evidenced by trypan blue exclusion and neutral red uptake. [3H]thymidine incorporation studies on CDT-treated cells were consistent with a blockage of cell proliferation without a direct inhibition of DNA synthesis. Although the progression of toxin action developed slowly, a 2-min exposure to CDT resulted in an irreversible development of toxicity. Together, our data indicate that CDT affects F-actin assembly within target cells and may interrupt the regulation or function of cell cycle-dependent events leading to cytokinesis.

Actins↗
PubMed2001

Induction of specific cytotoxic lymphocytes in mice vaccinated with Brucella abortus RB51.

A safe, more sensitive, nonradioactive, neutral red uptake assay was adopted to replace the traditional 51Cr release assay for detection of Brucella-specific cytotoxic T lymphocyte (CTL) activity. Our studies indicated that Brucella abortus strain RB51 vaccination of mice induced specific CTLs against both strain RB51- and strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. The antigen-specific cytotoxic activity was exerted by T lymphocytes but not by NK cells. CD3+ CD4+ T cells secreted the highest level of gamma interferon (IFN-gamma) and were able to exert a low but significant level of specific lysis of Brucella-infected macrophages. They also exerted a low level of nonspecific lysis of noninfected macrophages. In contrast, CD3+ CD8+ T cells secreted low levels of IFN-gamma but demonstrated high levels of specific lysis of Brucella-infected macrophages with no nonspecific lysis. These findings indicate that B. abortus strain RB51 vaccination of mice induces specific CTLs and suggest that CD3+ CD4+ and CD3+ CD8+ T cells play a synergistic role in the anti-Brucella activity.

Animals↗
PubMed2004

Attenuation of late-stage disease in mice infected by the Mycobacterium tuberculosis mutant lacking the SigF alternate sigma factor and identification of SigF-dependent genes by microarray analysis.

The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis DeltasigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type. In mice the growth rates of the DeltasigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection. At 8 weeks, the DeltasigF mutant persisted in the lung, while the wild type continued growing through 20 weeks. Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection. However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen. Intradermal vaccination of rabbits with the M. tuberculosis DeltasigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M. bovis BCG vaccination. Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase. Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the DeltasigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids. Examination of 5'-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX(18)GGGTAT. These results indicate that in the mouse the M. tuberculosis DeltasigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment. Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.

Animals↗
PubMed1977

Isolation and properties of two classes of low-density vesicles from Saccharomyces cerevisiae.

A mixture of small (0.43-mum diameter) and large (0.62-mum diameter) low-density vesicles from spheroplasts of Saccharomyces cerevisiae was fractionated by rate centrifugation in a gradient of 0 to 8% (wt/vol) Ficoll to yield fractions rich (90 to 95%) in small or large vesicles. The large, but not small, vesicles swelled when diluted into mannitol solutions containing less than 0.4 M mannitol. The pH-electrophoretic mobility curve of the large vesicles showed that they are probably enclosed in a phospholipid-protein membrane. The dyes neutral red and toluidine blue, accumulated into large vesicles by intact cells and spheroplasts, were largely lost from large vesicles when these were separated from stained spheroplasts. Sudan black III stained small and large vesicles, both classes of vesicle retaining the stain on separation. Fractions rich in large vesicles contained proportionately more phospholipid and less free sterols, diacylglycerols, and free fatty acids compared with those enriched in small vesicles. The two classes of vesicles contained about the same proportions of esterified sterols and triacylglycerols. The free fatty acids in both small and large vesicles were free from unsaturated fatty-acyl residues; diacylglycerols and triacylglycerols contained appreciable proportions of unsaturated fatty-acyl residues. Small vesicles were richer in lipase activity, whereas the larger vesicles contained greater beta-glucanase and alpha-mannosidase activities. Phospholipase activity could not be detected in any of the fractions.

Acid Phosphatase↗
PubMed1964

FORMATE--PYRUVATE EXCHANGE REACTION IN STREPTOCOCCUS FAECALIS. II. REACTION CONDITIONS FOR CELL EXTRACTS.

Oster, M. O. (A. & M. College of Texas, College Station), and N. P. Wood. Formate-pyruvate exchange reaction in Streptococcus faecalis. II. Reaction conditions for cell extracts. J. Bacteriol. 87:104-113. 1964.-In contrast to intact cells of Streptococcus faecalis, no stimulation of the formate-pyruvate exchange reaction was observed in cell extracts when yeast extract was added to the reaction mixture. A heated extract of Micrococcus lactilyticus, vitamin K(5), ferrous sulfate, and ferrous ammonium sulfate stimulated an active exchange by protecting the system from oxygen. Tetrahydrofolate, 2,3-dimercaptopropanol, and sodium sulfide provided partial protection, whereas ascorbate, glutathione, sodium hydrosulfite, ammonium sulfide, and sodium bisulfite gave insufficient protection or were inhibitory. Oxidation-reduction (O-R) indicators were not inhibitory and were used to estimate the O-R potentials of reaction mixtures. A potential at least as negative as -125 mv was estimated to be necessary to preserve or initiate formate-pyruvate exchange activity. The reaction operated over a narrow pH range when strict anaerobic conditions were not maintained but, when the system was suitably poised, the pH range was broader. The influence of high phosphate concentrations was less under strictly anaerobic conditions, and orthophosphate could be replaced by small amounts of pyrophosphate. Effect of temperature, time, and amount of extract is presented. Addition of reduced benzyl viologen and hydrogen-saturated palladium in the buffer during 8 hr of dialysis prevented inactivation of extracts. Recovery of activity could be obtained after ammonium sulfate treatment when a combination of palladium chloride, neutral red, and hydrogen bubbling were used.

Adenosine Triphosphate↗