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PubMed2001

Effects of burn with and without Escherichia coli infection in rats on intestinal vs. splenic T-cell responses.

OBJECTIVE: To evaluate the effect of burn injury with and without an Escherichia coliseptic complication on T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses in intestinal Peyer's patch and splenic T cells. DESIGN: Prospective, randomized, sham-controlled animal study. SETTING: University medical center research laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Rats were subjected to a 30% total body surface area, full skin thickness burn. Infection in rats was induced via intraperitoneal inoculation of E. coli, 10(9) colony forming units/kg, with or without a prior burn. MEASUREMENTS AND MAIN RESULTS: Rat Peyer's patch and splenic T lymphocytes were isolated by using a nylon wool cell purification protocol. T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses were measured after stimulation of cells with the mitogen, concanavalin A. T-cell proliferation was determined by measuring incorporation of (3)H-thymidine into T-cell cultures. Interleukin-2 production by T-cell cultures was measured by using enzyme-linked immunosorbent assay. Intracellular T-cell Ca2(+ )concentration, [Ca(2+)](i), was measured by the use of Ca(2+)-specific fluorescent label, fura-2, and its fluorometric quantification. [Ca(2+)](i) was also evaluated by the use of digital video imaging of fura-2 loaded individual T cells. T-cell proliferation and interleukin-2 production were suppressed substantially in both Peyer's patch and splenic T cells 3 days after either the initial burn alone or burn followed by the E. coli inoculation at 24 hrs after the initial burn. There seemed to be no demonstrable additive effects of E. coli infection on the effects produced by burn injury alone. The T-cell proliferation and interleukin-2 production suppressions with burn or burn-plus-infection insults were correlated with attenuated Ca(2+) signaling. E. coli infection alone suppressed T-cell proliferation in Peyer's patch but not in splenic T cells at 2 days postbacterial inoculation; E. coli infection had no effect on Peyer's patch or splenic T cells at 1 day postinjury. On the other hand, burn injury alone caused a substantial T-cell proliferative suppression at 2 days postburn in both Peyer's patch and splenic cells and a significant suppression in T-cell proliferation on day 1 postburn in Peyer's patch but not in the spleen. CONCLUSION: An initial burn injury suppressed T-cell proliferation at a level that it would not be further affected by a subsequent infection even if the infection by itself has the potential of suppressing T-cell proliferation. An earlier onset of T-cell suppression in Peyer's patch cells than in the spleen with burn could be attributable to an initial hypoperfusion-related intestinal mucosal tissue injury. Overall, our study supports the concept that burn injury per se can significantly suppress T-cell mediated immunity and that the intestine is an early tissue site of such suppression.

Analysis of Variance↗

PubMed2002

High expression of vascular endothelial growth factor predicts early recurrence and poor prognosis after curative resection for ductal adenocarcinoma of the pancreas.

INTRODUCTION AND AIMS: Only curative resection for pancreatic adenocarcinoma is related to a favorable prognosis, but the overall survival after surgery still remains poor, and early recurrence is frequently observed. Because recurrence is the limiting factor and the main cause of death after curative resection, the identification of markers that predict early postoperative recurrence is of paramount importance. Angiogenesis is essential for tumor growth and metastases; therefore, we set out to clarify whether vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) correlate with early recurrence and poor prognosis after curative resection. A second goal was to characterize the VEGF-producing cells and the subcellular distribution. METHODOLOGY: Seventy patients with ductal adenocarcinoma of the pancreas were studied after curative resection with a follow-up of at least 2 years. The MVD quantification was performed immunohistochemically with use of a monoclonal antibody to CD34. The VEGF expression was studied with use of polyclonal antibody. To detect the intracellular localization of specific VEGF mRNA sequences, nonisotopic in situ hybridization was performed. The correlations among VEGF expression and MVD, clinicopathologic parameters, and clinical outcome were then statistically analyzed. RESULTS: The VEGF immunoreactivity was 88.6%, and positive mRNA signals were obtained in the cytoplasm of carcinoma and endothelial cells in 81.4%. Furthermore, we observed tumor-associated macrophages close to infiltrating carcinoma cells. All endothelial cells showed positive immunoreactivity to the anti-CD34 antibody, and a median distribution of 85 vessels/x200 field was observed. A significant correlation (p < 0.05) was found between the MVD and the International Union Against Cancer (UICC) stage. Statistical analysis showed a significant correlation between VEGF expression and the height of MVD (p < 0.05). Kaplan-Meier analyses revealed that VEGF expression and MVD had a statistically significant correlation with survival after curative resection (p < 0.05). Furthermore, multivariate analysis indicated that VEGF expression is an independent prognostic marker for cancer recurrence within 8 months after curative surgery (p = 0.003). CONCLUSION: In pancreatic adenocarcinoma, the VEGF expression and the height of MVD are closely correlated, and both-rather than UICC stage and TNM classification (tumor size and nodal involvement)-are markers of prognostic relevance after curative resection. Furthermore, VEGF is a predictor of early recurrence after curative resection. The current study indicates that VEGF may promote the distribution of metastases, leading to early cancer recurrence and poor outcome.

Antigens, CD34↗

PubMed2000

Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells.

Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.

Animals↗

PubMedDate not supplied

Analysis of myocardial function in orthotopic cardiac allografts after prolonged storage in UW solution.

The need for a better organ preservative solution in heart transplantation is clear. At the same time, newer techniques in the assessment of cardiac function in the laboratory have made accurate load-independent quantification of myocardial preservation possible. Therefore a study was undertaken to evaluate left ventricular function in transplanted hearts after 14 hours of preservation in the intracellular lactobionate solution. Nine dogs were instrumented with ultrasonic dimension transducers, to measure left ventricular epicardial volume, and with micromanometers, to measure left ventricular pressure. Left ventricular wall volumes were determined from epicardial echocardiograms. To define the extent of organ injury resulting from the transplant procedure and cardiopulmonary bypass alone, four other animals were instrumented in a similar fashion, and left ventricular function was assessed after standard cardioplegic arrest and transplantation. The transplant procedures were performed with a warm ischemic period of 0.75 +/- 0.2 hours. In all experiments, data were collected before graft harvest and 1 hour after separation from cardiopulmonary bypass. Standard cardioplegic arrest and 2.4 +/- 0.1 hours of ischemia resulted in a decrease in left ventricular ejection fraction from 0.43 +/- 0.04 to 0.27 +/- 0.1 (37%) (p less than 0.01), a decrease in the slope of the stroke work/end-diastolic volume relationship from 15.4 +/- 7.9 to 7.9 +/- 2.0 erg X 10(4) (49%; p less than 0.01), and a decrease in the myocardial power output from 19.7 +/- 10.9 to 5.9 +/- 1.9 (70%; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1990

Quantification of alpha-fetoprotein and transferrin endocytosis by lymphoid cells using flow cytometry.

Alpha-fetoprotein (AFP) and transferrin (Tf) are serum proteins actively internalized by many growing cells through specific cell surface receptors. The intracellular pathways of AFP and Tf are very similar: both proteins enter the cells via coated pits and receptosomes, move to tubular elements of the transreticular portion of the Golgi and are recycled back to the cell surface and extracellular medium in native form. In the present work, the capacity of human lymphoid cells to bind AFP and Tf at 4 degrees C and to endocytose them at 37 degrees C was quantified by flow cytometry analysis on a FACS 440 using fluoresceinated derivatives of these proteins. The results obtained show that binding and internalization of AFP and Tf by lymphoid cells are saturable processes at either 4 degrees C or 37 degrees C. The method developed permits direct quantitative measurement of molecules bound to the cell surface or present within the cell.

Cell Line↗

PubMedDate not supplied

Lactate dehydrogenase quantification and isoenzyme distribution in physiological response to stress in red deer (Cervus elaphus).

As a contribution to the description of the physiological response to stress in red deer (Cervus elaphus), the potential use of the intracellular glycolytic enzyme, lactate dehydrogenase (LDH) and its isoenzyme LDH-5, as a marker of muscle damage was assessed. The distribution of LDH isoenzymes within red deer tissues was similar to that shown in other ruminants: tissue characterisation showed isoenzyme LDH-5 to be particularly associated with skeletal muscle. High plasma concentrations of creatine kinase, a muscle-specific enzyme, were associated with both high total LDH activity and the percentage of LDH-5 activity in deer undergoing a potentially stressful procedure, transportation followed by simulated abattoir lairage, which is further evidence of the specificity of the isoenzyme for skeletal muscle damage. The activity of LDH was not correlated with the plasma concentration of cortisol, a widely used physiological measure of psychological stress. This may have been due to different time courses for release and the fact that, in the present study, the immediate pre-collection procedures may have induced a short-term stress response.

Animals↗

PubMed2005

Determination of total ribonucleotide pool in plant materials by high-pH anion-exchange high-performance liquid chromatography following extraction with potassium hydroxide.

A new, improved method that only requires a potassium hydroxide extraction procedure is presented for the analysis of a full nucleotide pool in plant materials. Quantification was performed by high-pH anion-exchange chromatography (HPAEC) with UV detection after a potassium hydroxide extraction, and allowed the quantification of 13 linear ribonucleotides in a single run. The method has been validated by comparison of six extraction methods and also by measurement of the intracellular nucleotide levels of three plant species (cell cultures and leaves). The evolution of the nucleotide pool of Nicotiana tabacum cell culture during growth has also been measured, and showed an increase in the pool until the fifth day, where the growth rate reaches a maximum, after which a decrease was observed.

Anion Exchange Resins↗

PubMed1998

Differential mechanism for the cell surface sorting and agonist-promoted internalization of the alpha1B-adrenoceptor.

1. Alpha1B-adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist. Flow cytometry analysis with an anti-N-terminus-antibody (1B-N1-C, (Hirasawa et al., 1996)) facilitated the quantification of cell surface alpha1B-adrenoceptor. Also, the cellular distribution of alpha1B-adrenoceptors was visually monitored by immunocytochemical confocal microscopy. 2. Utilizing this combined approach, we have examined the molecular mechanism for cellular trafficking of alpha1B-adrenoceptors, including the process of sorting of the synthesized receptor protein to the cell surface, and the agonist-induced internalization. The two processes were separately examined by using alpha1B-adrenoceptor inducible DDT1MF-2 cells for the sorting process and CHO cells stably expressing alpha1B-adrenoceptors for the agonist-promoted internalization. 3. We examined the effects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), bafilomycin A1 (a specific inhibitor of the vacuolar proton pump) or hyperosmotic sucrose treatment (that may inhibit clathrin-mediated endocytosis) on these processes. 4. We found that the agonist-promoted internalization was blocked by cytochalasin D and mycalolide B, while the cell surface sorting process was specifically blocked by brefeldin A, indicating that the two processes involve different components of the cellular endocytic machinery. 5. The experimental approach as exemplified in this study would provide a valuable system to study further the molecular mechanism(s) of cellular trafficking of G protein-coupled receptors.

Adrenergic alpha-1 Receptor Agonists↗

PubMed2003

The type III secretion chaperone FlgN regulates flagellar assembly via a negative feedback loop containing its chaperone substrates FlgK and FlgL.

The type III secretion (TTS) chaperones are small proteins that act either as cytoplasmic bodyguards, protecting their secretion substrates from degradation and aggregation, facilitators of their cognate substrate secretion or both. FlgN has been previously shown to be a TTS chaperone for the hook-associated proteins FlgK and FlgL (FlgKL), and a translational regulator of the anti-sigma28 factor FlgM. Protein stability assays indicate that a flgN mutation leads to a dramatic decrease in the half-life of intracellular FlgK. However, using gene reporter fusions to flgK we show that a flgN mutation does not affect the translation of a flgK-lacZ fusion. Quantification of FlgM protein levels showed that FlgKL inhibit the positive regulation on flgM translation by FlgN when secretion of FlgKL is inhibited. Suppressors of the motility-defective phenotype of a flgN mutant were isolated and mapped to the clpXP and fliDST loci. Overexpression of flgKL on a plasmid also suppressed the motility defect of a flgN null mutant. These results suggest that FlgN is not required for secretion of FlgKL and that FlgN typifies a class of TTS chaperones that allows for the minimal amount of their substrates expression required in the assembly process by protecting the substrate from proteolysis. Our data leads us to propose a model in which the interaction between FlgN and FlgK or FlgL is a sensing mechanism to determine the stage of flagellar assembly. Furthermore, the interaction between FlgN and FlgK or FlgL inhibits the translational regulation of flgM via FlgN in response to the stage of flagellar assembly.

Adenosine Triphosphatases↗

PubMed2003

Transcriptional and translational regulation of the splicing isoforms of the growth hormone receptor by glucocorticoids.

Glucocorticoids exert multiple effects on the growth hormone IGF-I axis. The GH receptor is expressed as an active, full-sequence molecule and a truncated, inactive one that lacks the intracellular signaling domain. We used the HuH7 human hepatoma cell line to investigate the effect of glucocorticoids on growth hormone receptor mRNA and protein expression. cDNA quantification was performed by Real Time Quantitative PCR. Growth hormone receptor expression at the protein level was studied by fluorescence-activated cell sorter analysis using specific polyclonal antibodies raised against the two isoform unique C-terminal sequences. Cells were treated with pharmacologic doses of dexamethasone (Dex 10 -7 - 10 -5 M) to assess its acute (1 hour or overnight) and chronic effects (7 days). Dex induced a dose-dependent increase of both the full (427 %) and truncated (180 %) mRNAs. At the protein level, Dex upregulated the full sequence more (183 %) than the truncated (126 %) protein. For a better understanding of this regulation system, cells were incubated with Dex 10 -6 M for 24 h in the absence or presence of a transcriptional inhibitor, actinomycin D, or a translational inhibitor, cycloheximide. Actinomycin D had no effect on Dex-induced upregulation, while cycloheximide blocked the truncated mRNA but not the full sequence mRNA upregulation, suggesting that this effect of glucocorticoids is a post-transcriptional event. After 7 days of chronic treatment, Dex induced a dose-dependent downregulation of the active receptor without any changes in the expression of the truncated isoform either at the mRNA or protein levels. We conclude that short-term glucocorticoid treatment results in an enhancement of the growth hormone receptor expression, while long-term treatment has a suppressive effect, through both transcriptional and translational mechanisms ultimately influencing both isoforms of the receptor.

Alternative Splicing↗

PubMed2006

Calcitonin gene-related peptide stimulates human villous trophoblast cell differentiation in vitro.

Calcitonin gene-related peptide (CGRP) is a multifunctional peptide present in both maternal and fetal circulations in pregnancy. Its receptors have been identified on human trophoblast cells, but the role of CGRP in trophoblast differentiation remains unknown. This study was designed to determine the effect of CGRP on the differentiation of villous trophoblasts isolated from normal human term placentae. The morphological and functional differentiation of the trophoblast cells were assessed by desmosomal protein immunofluorescent staining and the quantification of hCG, estrogen and progesterone secretion. Results showed that (i) exposure of villous trophoblast cells to CGRP led to a dose-dependent increase in intracellular cyclic adenosine monophosphate (cAMP) accumulation; (ii) immunofluorescent staining with antidesmosomal antibody was identified at the boundaries between aggregated cytotrophoblast cells, and these stainings disappeared when cells fused to form syncytiotrophoblast cells; (iii) the formation of multinucleated syncytiums in primary cultured cytotrophoblasts was stimulated by CGRP as evidenced by the changes in antidesmosomal staining; (iv) CGRP increases trophoblast hCG secretion in a time- and dose-dependent manner, and this secretion was blocked by CGRP antagonist, CGRP(8-37), and (v) both 17beta-estradiol (E(2)) and progesterone concentrations in the culture medium were increased by CGRP, and these increases were dose dependent. These observations suggest that CGRP may be involved in the morphological and functional differentiation of trophoblast cells, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.

Calcitonin Gene-Related Peptide↗

PubMed1992

Long-term in-vitro treatment of human growth hormone (GH)-secreting pituitary adenoma cells with octreotide causes accumulation of intracellular GH and GH mRNA levels.

OBJECTIVE: We studied the effects of long-term in-vitro exposure of human GH secreting pituitary adenoma cells to octreotide on GH release, intracellular GH concentrations and GH messenger ribonucleic acid (mRNA) levels. DESIGN: Human GH-secreting pituitary adenoma cells were cultured for periods from 4 days up to 3 weeks without or with octreotide (10 nM) and/or bromocriptine (10 nM). The effects of these drugs were measured on GH release, intracellular GH concentrations and intracellular GH mRNA levels. PATIENTS: Thirteen patients with GH-secreting pituitary adenomas were studied. Twelve patients were untreated, one had been pretreated with octreotide (12 weeks, 3 x 100 micrograms daily). MEASUREMENTS: GH, PRL, alpha-subunit and IGF-I concentrations in plasma, media and cell extracts were determined by immunoradiometric or radioimmuno-assays. GH mRNA levels were determined by automatic quantification of grain numbers in individual adenoma cells. RESULTS: Incubation of the adenoma cells for 4 days with 10 nM octreotide induced a dose-dependent inhibition of GH release and a parallel increase (increase varying between 124 and 617% of control) in the intracellular GH levels was observed in six of seven adenomas. In addition, bromocriptine, when effective in inhibiting GH release by the adenomas, also induced an increase in intracellular GH levels. Even after 3 weeks of exposure to 10 nM octreotide in vitro there was a statistically significant increase in intracellular GH levels (between 191 and 923% of control). Withdrawal of octreotide after 6 days of incubation resulted in a lowering of intracellular GH levels to control values, showing that the octreotide-induced increase in intracellular GH is reversible. In a 96-hour incubation with 10 nM octreotide, GH mRNA levels were increased in two, and slightly decreased in one of the three adenomas tested. This effect was time dependent in that there was no significant effect of 10 nM octreotide on GH mRNA levels in a 24-hour incubation. CONCLUSIONS: (1) Long-term in-vitro exposure of GH-adenoma cells to octreotide causes an increase in intracellular GH levels in the majority of the adenomas, probably because of an increase in GH mRNA levels in the adenoma cells; and (2) this considerable increase in intracellular GH levels may be one of the explanations for the relatively poor effect of octreotide on tumour shrinkage in patients with GH-secreting pituitary adenomas.

Adenoma↗

PubMed1981

Colloidal gold : a cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy.

Gold sols are orange to violet, display electron dense properties and are capable of strong emission of secondary electrons. These properties enable gold particles to be used as specific markers in microscopy both at the low and high resolution level (light and fluorescent microscopy, scanning and transmission electron microscopy). Monodisperse colloidal gold can be produced by several methods in a size range of 5 nm to 150 nm. As a consequence, the gold method is well suited for multiple marking experiment at the high resolution level. Since gold markers bind non-specifically to a very low extent, the technique has found application in TEM for marking intracellular components on thin sections. Both the one step and the two step marking procedures have been utilized in the various modes of microscopy. Under appropriate conditions, gold particles can be labelled with a variety of macromolecules (polysaccharides, glycoproteins, proteins, lectins, antibodies), presumably through a noncovalent binding process. Generally the probes acquire the specific activity of the adsorbed macromolecule and their stability upon storage is good. A number of factors which influence the adsorption process are discussed in relation to the more general problems of adsorption of macromolecules onto metallic surfaces. The stability of gold markers is also best understood by the DLVO theory for disperse systems. The preparation, labelling, stabilization, stability and binding characteristics of gold markers are reviewed. Since the binding of gold probes to cell surfaces is primarily determined by the size of the particle, several problems related to steric hindrance and quantification of the method are also discussed. The advantages of the method over others are compared. The different modes of microscopy and the several gold methods available for marking cell surface and intracellular components are illustrated by micrographs.

Adsorption↗

PubMed2007

Expression of rotavirus NSP4 alters the actin network organization through the actin remodeling protein cofilin.

Rotavirus is a major cause of infantile gastroenteritis with a multifactorial pathogenesis. As with many other pathogens, rotavirus infection and replication leads to rearrangement of the cytoskeleton with disorganization of cytoskeletal elements such as actin and cytokeratin through a calcium-dependent process that has not been fully characterized. The rotavirus enterotoxin NSP4, shown previously to elevate intracellular calcium levels when added exogenously as well as when expressed intracellularly, is a key player in intracellular calcium regulation during rotavirus infection. Here, we investigated the role NSP4 may play in actin rearrangement. Expression of NSP4 fused to enhanced green fluorescent protein (NSP4-EGFP), but not expression of EGFP alone, caused stabilization of long cellular projections in fully confluent HEK 293 cells. Cells expressing NSP4-EGFP for 24 h were also resistant to cell rounding induced by cytochalasin D. Quantification of filamentous actin (F-actin) content by using rhodamine-conjugated phalloidin and flow cytometry showed an elevated F-actin content in NSP4-EGFP-expressing and rotavirus-infected cells in comparison with that in nonexpressing and noninfected cells. Normalization of intracellular calcium levels prevented alterations of F-actin content. Observed changes in F-actin amounts correlated with the increased activation of the actin-remodeling protein cofilin. These calcium-dependent actin rearrangements induced by intracellular NSP4 expression may contribute to rotavirus pathogenesis by interfering with cellular processes dependent on subcortical actin remodeling, including ion transport and viral release.

Actin Depolymerizing Factors↗

PubMed2001

Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies.

Bovine Reissner's fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen-mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs recognizing the bovine RF- and SCO-glycoproteins in solutions (ELISA) as well as in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non-mammalian species. The different types of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti-RF serum. It is concluded that all Mabs raised in our laboratory are directed against non-repeated sequences of RF-glycoproteins that have not been conserved in vertebrate phylogeny.

Animals↗

PubMedDate not supplied

An in vitro model for toxicological investigations of environmental neurotoxins in primary neuronal cell cultures.

Currently, most neurotoxicological investigations are still conducted using various animal models (e.g. chickens, rodents). In this report, alternative strategies of testing were examined to detect the neurotoxic potency of foreign compounds. Primary neuronal cell cultures from fetal rats are already an accepted model for mechanistic and pharmacological studies in drug research. Their suitability for neurotoxicological studies was examined by using industrial model compounds, which are well-known inductors of neuropathies: acrylamide, hexachlorophene, paraquat, n-hexane, and its neurotoxic metabolites acetylaceton and 2,5-hexandione. As a control compound, the nonneurotoxic solvent n-heptane was used. General cytotoxicity and the intracellular content of glial fibrillary acid protein, neuron-specific enolase, and neurofilaments were measured. n-Heptane induced an acute cytotoxicity and acrylamide and 2,5-hexandione produced a delayed cytotoxicity in primary neuronal cells, whereas the others showed no cytotoxic potency in the tested concentration range. These results were in agreement with the quantification of neurons by neuron-specific enolase. In contrast, with the exception of acetylaceton, glia cells were significantly affected by all neurotoxins at the later time. Signs of axonopathies were demonstrated for acrylamide, n-hexane and its metabolites, as well as for hexachlorophene and paraquat in vitro, by determining the intracellular neurofilament level. Therefore, the determination of cell-specific end points is necessary to detect the neurotoxic potency and quality of a compound, whereas the cytotoxicity assay limited the tested concentration range.

Acrylamide↗

PubMed2001

Effects of trimethoprim and co-trimoxazole on the morphology of Listeria monocytogenes in culture medium and after phagocytosis.

The purpose of this study was to compare the extra- and intracellular activity of antifolates on Listeria monocytogenes. The fortuitous discovery of elongated bacteria in response to trimethoprim revealed a novel effect on the morphology of Listeria in cell culture medium and after phagocytosis. This phenomenon permitted the quantification of trimethoprim activity, revealing comparable activity intra- and extracellularly. Subinhibitory concentrations of trimethoprim resulted in bacterial elongation, which was reversed after removal of trimethoprim. We attribute this effect of trimethoprim to an inhibition of cell wall synthesis and/or cell separation of Listeria.

Animals↗

PubMed1993

Morphometric analysis of mucous granule depletion and replenishment in rat colon.

Computer-assisted morphometric analysis was used to quantify mucous granule discharge and the subsequent replenishment of secretory granules in the rat colon following cholinergic challenge. Within 5 min of stimulation (250 micrograms/kg carbachol, subcutaneous), the volume of intracellular mucous granules decreased to 61.5% of the control. The apical plasma membranes of goblet cells in the midcrypt region became deeply cavitated, indicating that they had accelerated mucous granule secretion by a process of compound exocytosis. Goblet cells at the base of the crypt rarely showed cavitated apical membranes but clearly became depleted of intracellular mucous granules. At 60 min after stimulation, cavitated profiles were very rare (< or = 0.4%) but intracellular stores of mucous granules were still significantly depressed (54.7% of control). By 4 hr after stimulation, mucous granule stores recovered to 94.9% of control levels. Morphometric quantification was found to be a reliable and sensitive measure of recent goblet cell secretory activity.

Animals↗