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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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria.

Here, we describe a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria. The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometic measurement of enzymatic activity. As a model system, we used virulent phage typing and cell-marker enzyme activity (beta-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655). Filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 h. In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage.

Bacteria↗

Subcellular compartmentation of prolactin in rat lactotrophs.

The intracellular localization of different molecular forms of prolactin was studied in various experimental models covering a wide range of secretory states. By correlating electron microscopy, morphometry and quantification of monomeric (small) and polymeric (big) prolactin after differential extraction procedures, big prolactin was found stored in secretory granules while small prolactin was loosely associated with all organelles involved in hormone synthesis and processing. No correlation with levels of lactotrophic secretory activity was detected by either the number of secretory granules or prolactin content in lactotrophs.

Animals↗

Deoxynucleotide-interconverting enzymes and the quantification of deoxynucleoside triphosphates in mammalian cells.

We have demonstrated that methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in haematopoietic cells are described which provide sensitivity to measure 0.1-40pmol of dATP, dTTP and dGTP, and 1.0-40pmol of dCTP. The extraction and assay give linear results with (2.5-15)x10(6) nucleated cells and (0.1-1.5)x10(9) red blood cells. Under these conditions, extracts equivalent to ~0.5x10(6) nucleated haematopoietic cells catalyse the phosphorylation of 0-8% of dNDP and dNMP standards to dNTP and incorporate them into deoxynucleotide polymer under circumstances where 100% of an equimolar dNTP standard would be incorporated. By contrast, extracts of 0.4x10(6) HeLa cells totally converted dADP, dTDP and dGDP into dNTP with subsequent polymerization. Conversion of dCDP was somewhat less efficient. The results demonstrate conclusively that the activities of deoxynucleotide interconverting enzymes differ in different types of human cells. They can interfere with assay of nucleotides, but may not do so in many types of cell extracts. In particular, dNTP concentrations can be measured in human haematopoietic cells after extraction with 60% (v/v) methanol and are not artificially elevated by deoxynucleotide interconversions. It is apparent that extraction and assay procedures for measurement of dNTP species should be analysed for each cell type in order to minimize contaminating enzyme activities and ensure accuracy of dNTP quantification.

Cell Line↗

Metabolic physiology of aroma-producing Kluyveromyces marxianus.

Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from L-phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2-phenylethanol/mol L-phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC-MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring-labelled ((13)C(6)) L-phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2-phenylethanol and 2-phenylethylacetate stem exclusively from the supplied L-phenylalanine, whereas glucose was not converted into these products because of efficient feed-back inhibition of prephenate dehydratase in the L-phenylalanine biosynthetic pathway. It could be further shown that the supplied L-phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of (13)CO(2) in the exhaust gas provided clear evidence for catabolic breakdown of L-phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L-phenylalanine revealed a significant loss of L-phenylalanine into catabolic and anabolic pathways. While 73.3% of L-phenylalanine was converted into 2-phenylethanol or 2-phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L-phenylalanine via hydroxylation to L-tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2-phenylethanol-producing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts.

Biotechnology↗

Quantification of human dopamine D2s receptor interactions with G alpha(i,1,2)- and G alpha(o)-proteins.

A simple and rapid in vitro method for qualitative and quantitative estimation of the G alpha-subunits interaction with the third intracellular loop of human D2s dopamine receptor has been developed. For this purpose, D2s-CL3 was cloned in pGEX-2T vector and expressed in E. coli BL21 DE3 as a fusion protein with glutathione-S-transferase (D2s-CL3-GST). The resulting soluble construct was purified by affinity chromatography on glutathione-Sepharose. G alpha-subunits were expressed and purified as His-tagged proteins. For the assay of G alpha/D2s-CL3-GST interactions, varying concentrations of pure His-tagged G alpha-proteins were immobilized on His-Bind Resin and titrated with D2s-CL3-GST fusion protein. G alpha/D2s-CL3-GST interactions were quantified by GST activity determination assay. It was shown that the fusion protein interacts specifically with different G alpha proteins, especially with G alpha(i) proteins. Based on saturation binding analyses, Kd values were determined revealing the highest affinity of His-G alpha(i,2) binding to the fusion protein. The affinities for G alpha(i)/D2s-CL3-GST protein interactions estimated in this way were in nanomolar range of concentrations.

Escherichia coli↗

[Ca(2+)](i) oscillations regulate type II cell exocytosis in the pulmonary alveolus.

Pulmonary surfactant, a critical determinant of alveolar stability, is secreted by alveolar type II cells by exocytosis of lamellar bodies (LBs). To determine exocytosis mechanisms in situ, we imaged single alveolar cells from the isolated blood-perfused rat lung. We quantified cytosolic Ca(2+) concentration ([Ca(2+)](i)) by the fura 2 method and LB exocytosis as the loss of cell fluorescence of LysoTracker Green. We identified alveolar cell type by immunofluorescence in situ. A 15-s lung expansion induced synchronous [Ca(2+)](i) oscillations in all alveolar cells and LB exocytosis in type II cells. The exocytosis rate correlated with the frequency of [Ca(2+)](i) oscillations. Fluorescence of the lipidophilic dye FM1-43 indicated multiple exocytosis sites per cell. Intracellular Ca(2+) chelation and gap junctional inhibition each blocked [Ca(2+)](i) oscillations and exocytosis in type II cells. We demonstrated the feasibility of real-time quantifications in alveolar cells in situ. We conclude that in lung expansion, type II cell exocytosis is modulated by the frequency of intercellularly communicated [Ca(2+)](i) oscillations that are likely to be initiated in type I cells. Thus during lung inflation, type I cells may act as alveolar mechanotransducers that regulate type II cell secretion.

Animals↗