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PubMed1985

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.

Blood Platelets↗

PubMed2002

Design and applications of methods for fluorescence detection of iron in biological systems.

Fluorescence metalosensors provide a means to detect iron in biological systems that is versatile, economical, sensitive and of a high-throughput nature. They rely on relatively high-affinity iron-binding carriers conjugated to highly fluorescent probes that undergo quenching after metal complexation. Metal specificity is determined by probes containing either an iron-binding moiety of high affinity (type A) or of relatively lower affinity (type B) used in combination with a strong specific iron chelator. Due to the heterogeneous nature of biological systems, the apparent metal-binding affinity and complexation stoichiometry ought to be specifically defined. Fluoresceinated moieties coupled to metal-binding cores detect Fe at sub-micromolar concentrations and even sub-microlitre volumes (i.e. cells). Although an ideal probe should also be specific for a particular oxidation state of iron, in physiological conditions that property might be difficult to attain. Quantification of labile iron in cells has relied on the ability of permeant iron chelators to restore the fluorescence of probes quenched by intracellular Fe. Modern design of probes aims to (a) improve probe targeting to specific cell compartments and (b) create probes that respond to metal binding by signal enhancement.

Calibration↗

PubMed2002

Distribution and quantification of polyethylenimine oligodeoxynucleotide complexes in human skin after iontophoretic delivery using confocal scanning laser microscopy.

Iontophoresis may be a potentially useful technique for the delivery of oligonucleotides into the skin. To enhance intracellular uptake during iontophoresis, we investigated the dermal delivery of oligodeoxynucleotides (ODN) as a polyelectrolyte complex with polyethylenimine (PEI). Perpendicular cross-sectioning was performed to visualize and quantify the penetration properties of double labeled PEI/ODN complexes across full thickness human skin. Due to the net positive charge of the complexes, anodal iontophoresis was expected to enhance skin delivery by electrorepulsion compared to passive diffusion. Confocal laser scanning microscopy demonstrated that non-complexed ODN could penetrate the skin after 1 h of cathodal iontophoresis but not by passive diffusion or anodal iontophoresis. However, extensive degradation occurred as documented by a dramatic decrease of fluorescence intensity within viable skin tissue after 10 h. Anodal iontophoresis of the complexes led to a deep penetration of both the TAMRA-labeled ODN and the Oregon Green-labeled PEI. A constant increase in fluorescence indicated a protective effect of the polymer against nuclease degradation. Co-localization of red and green fluorescence was noted within numerous nuclei of epidermal keratinocytes. In contrast, passive diffusion of the complexes did not lead to successful uptake into keratinocytes and was limited to the stratum corneum. Complexation of ODN by PEI, therefore, seems to be a promising method to enhance both the transport of charged complexes into the skin and to facilitate intracellular uptake, which may potentially be useful for the local treatment of skin diseases using ODN.

Drug Carriers↗

PubMed1992

Glucose regulates its transport in L8 myocytes by modulating cellular trafficking of the transporter GLUT-1.

The effect of culture conditions simulating hypo- and hyper-glycaemia on glucose transport and on the subcellular localization of the glucose transporter GLUT-1 was studied in L8 myocytes. Incubation of the cells with 20 mM-glucose for 25 h decreased the rate of 2-deoxy-D-[3H]glucose (dGlc) uptake to 0.106 +/- 0.016 nmol/min per 10(6) cells compared with 0.212 +/- 0.025 in cells maintained at 2 mM-glucose (final glucose concentrations at the end of the incubation period were 16-17 mM and 0.7-1.0 mM respectively). An additional 5 h incubation of these cells with medium containing the opposite glucose concentration (i.e. change from 17 mM to 1 mM and from 1 mM to 17 mM) increased the transport rate to 0.172 +/- 0.033 nmol/min per 10(6) cells in cultures initially conditioned at high glucose, and decreased the transport to 0.125 +/- 0.029 in those conditioned at low glucose. Plasma-membrane- and microsomal-membrane-enriched fractions were prepared from these cells for [3H]cytochalasin B (CB) binding and Western-blot analysis with antibodies against GLUT-1 and GLUT-4. A decrease in glucose concentration increased the number of D-glucose-displaceable CB-binding sites and GLUT-1 protein in the plasma-membrane fraction to the same extent as the increase in dGlc transport. Under downregulatory conditions, the lower dGlc-transport capacity could be accounted for by a decreased number of transporters in the plasma membrane of the cells. No apparent modification of the intrinsic activity of the glucose transporters was observed in up- or down-regulated cells. Under downregulatory conditions, the CB-binding data indicated a large increase in the number of transporters in the intracellular membranes of the myocytes. Western blots of the same membranes also indicated an increase in GLUT-1 content. However, the interaction of the intracellular GLUT-1 protein with the polyclonal antibodies was much weaker than that of the plasma-membrane-associated GLUT-1. The GLUT-4 concentration was too low to permit quantification in membrane fractions. Our findings suggest that autoregulation of glucose transport in L8 myocytes is accompanied by parallel changes in the number of GLUT-1 transporters in the plasma membrane, and that the rate of transporter degradation may be augmented in the upregulated myocytes. These glucose-induced changes are fully reversible.

Animals↗

PubMed1996

High-performance liquid chromatographic methods for the quantification of tetrahydrobiopterin biosynthetic enzymes.

Tetrahydrobiopterin is a cofactor in hydroxylation reactions, including phenylalanine 4-monooxygenase, tyrosine 3-monooxygenase, tryptophan 5-monooxygenase, alkyl glycol ether monooxygenase and nitric oxide synthase. Determination of its biosynthesis is carried out to diagnose inherited diseases leading to partial defects in tetrahydrobiopterin synthesis. In addition, tetrahydrobiopterin synthesis is induced by proinflammatory cytokines, and intracellular levels of tetrahydro-biopterin in many cases limit the activity of tetrahydrobiopterin-dependent reactions, such as nitric oxide synthase in intact cells. Biosynthesis of tetrahydrobiopterin from guanosine 5'-triphosphate (GTP) requires the action of three enzymes, GTP-cyclohydrolase I (E.C. 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase (EC, 4.6.1.10) and sepiapterin reductase (E.C. 1.1.1.153). Methods for quantification of biopterin and related pteridines in biological matrices by HPLC and application of these for determining the activity of the three tetrahydrobiopterin biosynthetic enzymes are reviewed in this article.

Alcohol Oxidoreductases↗

PubMed1999

Tumour phospholipid metabolism.

Following the impetus of early clinical and experimental investigations, in vivo and in vitro MRS studies of tumours pointed in the eighties to the possible significance of signals arising from phospholipid (PL) precursors and catabolites as novel biochemical indicators of in vivo tumour progression and response to therapy. In the present decade, MRS analyses of individual components contributing to the 31P PME (phosphomonoester) and PDE (phosphodiester) resonances, as well as to the 1H 'choline peak', have reinforced some of these expectations. Moreover, the absolute quantification of these signals provided the basis for addressing more specific (although still open) questions on the biochemical mechanisms responsible for the formation of intracellular pools of PL derivatives in tumours, under different conditions of cell proliferative status and/or malignancy level. This article is aimed at providing an overview on: (a) quantitative MRS measurements on the contents of phosphocholine (PCho), phosphoethanolamine (PEtn) and their glycerol derivatives ģlycerol 3-phosphocholine (GPC) and glycerol 3-phosphoethanolamine (GPE)[ in human tumours and cells (with particular attention to breast and brain cancer and lymphomas), as well as in normal mammalian tissues (including developing organs and rapidly proliferating tissues); (b) possible correlations of MRS parameters like PEtn/PCho and PCho/GPC ratios with in vitro cell growth status and/or cell tumorigenicity; and (c) current and new hypotheses on the role and interplay of biosynthetic and catabolic pathways of the choline and ethanolamine cycles in modulating the intracellular sizes of PCho and PEtn pools, either in response to mitogenic stimuli or in relation to malignant transformation.

Animals↗

PubMed2001

Cell function-based flow cytometry.

Flow cytometry is increasingly used to assess the functional status of leukocytes, and practically all aspects of their life (and death) are accessible to flow cytometric study. Together with familiar features of flow cytometry, such as multiparameter immunophenotyping, cell function-based flow cytometry has provided many new insights into the relationships among lymphocyte cell surface features; intracellular processes, such as cytokine production and protein phosphorylation; and the functional status of lymphocytes in a variety of human diseases. Direct visualization and quantification of antigen-specific T cells using major histocompatibility complex (MHC)-peptide tetramer technology, in combination with functional assays, has provided the means to study specific T-cell subsets of interest. Even early in its development, this technology already has offered a better understanding of basic and clinical immunology and has invited reassessment of several long-standing immunologic concepts. Semin Hematol 38:169-178. This is a US government work. There are no restrictions on its use.

Animals↗

PubMed2003

A new histamine measurement system employing a confocal laser microscope and cultured human dermal microvascular endothelial cells.

BACKGROUND: Histamine causes the endothelial cells of blood vessels to contract. H(1) receptor antagonists block histamine from binding to the H(1) receptor, suppressing edema. But it is unknown whether they bring about similar effects in microvascular endothelial cells in the human skin. OBJECTIVE: We devised a series of experiments to measure changes in Ca(2+) concentration within human dermal microvascular endothelial cells stimulated with histamine using a confocal laser microscope. Within a distinct concentration range, the observed changes in Ca(2+) concentration were proportional to the amount of histamine administered, raising the possibility that trace amounts of histamine and the amount of histamine binding to H(1) receptors could be quantified. METHODS: An experiment was conducted in which the H(1) receptor-antagonistic actions of eight antihistamines and anti-allergy drugs with antihistamine properties were quantitatively evaluated using a system for performing the above quantification. Each evaluated drug was preloaded within the range 10(-9)-10(-3) M, histamine 10(-6) M was added, and changes in the intracellular concentration of Ca(2+) were noted so as to evaluate the drugs' antagonism of the H(1) receptor. RESULTS: Within the measurable range of the system, epinastine was the most effective of the drugs tested in inhibiting changes in Ca(2+) concentration in cells stimulated with histamine. CONCLUSION: Our system can be used to quantitatively measure and compare the actions of H(1) receptor antagonists on dermal microvascular endothelial cells, providing valuable information for dermatologists.

Anti-Allergic Agents↗

PubMed1994

Sphingosine kinase from Swiss 3T3 fibroblasts: a convenient assay for the measurement of intracellular levels of free sphingoid bases.

Sphingoid bases are important for diverse cellular processes. This paper describes the development of a simple and rapid enzymatic method to quantify mass levels of free long-chain sphingoid bases in cellular lipid extracts. The assay is based on the ability of sphingosine kinase from Swiss 3T3 fibroblasts to phosphorylate sphingoid bases, predominantly sphingosine. To quantitatively determine cellular levels of sphingosine or other long-chain sphingoid bases, it was necessary to solubilize cellular lipids by sonication and to generate standard curves in the presence of lipid extracts containing at least 50 nmol of phospholipids. The assay conditions were optimized to allow quantification of sphingosine over a broad range from 25 to 1,000 pmol. Using this method we were able to obtain reproducible measurements of free sphingoid bases in various cell types and to detect increases in intracellular sphingoid base levels after treatment with exogenous sphingosine or stimulation with fetal bovine serum.

3T3 Cells↗

PubMed2001

Invasion and persistent intracellular colonization of erythrocytes. A unique parasitic strategy of the emerging pathogen Bartonella.

The expanding genus Bartonella includes zoonotic and human-specific pathogens that can cause a wide range of clinical manifestations. A productive infection allowing bacterial transmission by blood-sucking arthropods is marked by an intraerythrocytic bacteremia that occurs exclusively in specific human or animal reservoir hosts. Incidental human infection by animal-adapted bartonellae can cause disease without evidence for erythrocyte parasitism. A better understanding of the intraerythrocytic lifestyle of bartonellae may permit the design of strategies to control the reservoir and transmittable stages of these emerging pathogens. We have dissected the process of Bartonella erythrocyte parasitism in experimentally infected animals using a novel approach for tracking blood infections based on flow cytometric quantification of green fluorescent protein-expressing bacteria during their interaction with in vivo-biotinylated erythrocytes. Bacteremia onset occurs several days after inoculation by a synchronous wave of bacterial invasion into mature erythrocytes. Intracellular bacteria replicate until reaching a stagnant number, which is sustained for the remaining life span of the infected erythrocyte. The initial wave of erythrocyte infection is followed by reinfection waves occurring at intervals of several days. Our findings unravel a unique bacterial persistence strategy adapted to a nonhemolytic intracellular colonization of erythrocytes that preserves the pathogen for efficient transmission by blood-sucking arthropods.

Animals↗

PubMed1998

Interleukin-4 and interleukin-10 are chondroprotective and decrease mononuclear cell recruitment in human rheumatoid synovium in vivo.

We used the severe combined immunodeficient (SCID) mouse model to assess the effect of interleukin-4 (IL-4) or IL-10 injection on cartilage degradation and mononuclear cell (MNC) recruitment to human rheumatoid synovium in vivo. Human rheumatoid synovium and cartilage from five rheumatoid arthritis patients, obtained after joint replacement surgery, were engrafted subcutaneously to 6-8-week-old SCID CB17 mice. Synovial tissues were injected with recombinant human IL-4 (rhIL-4, 100 ng; rhIL-10, 100 ng), both cytokines, or tumour necrosis factor-alpha (TNF-alpha) (1000 U), or phosphate-buffered saline twice a week for 4 weeks. The graft was removed and immunochemical analysis was carried out to assess intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression. Moreover, cartilage degradation was assessed through the quantification of the erosion surface on a computerized image of the engrafted cartilage at high power view. MNC recruitment in the synovial tissue was determined by labelling blood MNC with indium-111 before their intraperitoneal injection. The activity obtained in the region of the graft were determined with a gamma camera 72 hr postinjection. The results are expressed as a percentage of initial injected activity. After 4 weeks we observed a decrease of cartilage area in controls (77 +/- 8%), inhibited after injection of IL-4, IL-10, or both cytokines (90 +/- 3%, 89.1 +/- 4%, 89.2 +/- 5% respectively), and 57 +/- 17% after TNF-alpha injection. The % MNC activity in the graft decreased to 77 +/- 81% (NS), 9 +/- 4% (P < 0.003) and 19 +/- 6% (P < 0.007) compared with untreated synovial tissue after treatment with IL-4, IL-10, or both cytokines, respectively. Moreover, IL-10 but not IL-4 decreased the expression of ICAM-1 but not VCAM-1 or E-selectin by synovial cells. These results suggest that IL-10 and IL-4 could have chondroprotective properties, and that IL-10 but not IL-4 inhibits MNC traffic towards the synovial tissue efficiently.

Animals↗

PubMed2002

Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycaemic conditions.

The neuronal effects of glucose deficiency on amino acid metabolism was studied on three-dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone-specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose-deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.

Amino Acids↗

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↗