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PubMed1988

ESTA: a bioassay system for the determination of the potencies of hormones and antibodies which mimic their action.

A bioassay system named ESTA (eluted stain assay) has been developed to measure hormones and antibodies which mimic their action. It is derived from approaches used for cytochemical bioassays. Unlike the latter which use tissue segments or sections, ESTA is based upon uniform microcultures of target cells maintained in microtitre plates. Direct elution of the cytochemical stain from these microcultures into the wells of the microtitre plates permits rapid quantification with a microtitre plate reader. We describe ESTA systems for GH, prolactin, thyroid stimulators and human chorionic gonadotrophin which utilize the reduction of a tetrazolium salt to a formazan by intracellular dehydrogenase as the cytochemical system. These provide examples of ESTA systems in which the assay signal depends solely upon an increase in cell number in response to the hormone, or in which there is additional enzymic amplification.

Animals↗

PubMed1991

Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry.

Precise estimates of the frequency of NK- and LAK-target conjugates were obtained by two-color flow cytometry using hydroethidine and calcein as intracellular labels for target cells and effector cells, respectively. These two dyes can easily be used with a standard single-laser flow cytometer with excellent signal separation and dye retention. Hydroethidine labeling did not alter target susceptibility, and calcein labeling did not significantly alter NK function. Excellent agreement was obtained between this flow cytometric method and visual estimation of the frequency of fresh or IL-2-activated human lymphocytes that form conjugates with K-562 target cells. The percentage of cloned NK or LAK cells that form conjugates with K-562 target cells was dependent on the E:T ratio, with extrapolated maximum conjugate frequencies (alpha max) of 40-50%. However, the frequency of lymphocytes forming conjugates with K-562 cells did not closely correlate with the cytolytic activity of a given lymphocyte population. This two color flow cytometric method employing a pair of fluorochromes that do not modify cell membranes or alter cell function in cytotoxicity assays should facilitate further studies of mechanisms involved in the initial stages of target cell recognition by NK and LAK cells.

Cell Survival↗

PubMed1986

Secretory granules of heparin-containing rat serosal mast cells also possess highly sulfated chondroitin sulfate proteoglycans.

Rat serosal mast cells, which synthesize only heparin proteoglycans as detected by intrinsic labeling with [35S]sulfate, were analyzed for the presence of intracellular chondroitin sulfate proteoglycans by chemical and immunochemical means. Rat serosal mast cells of greater than 99% purity were treated with Zwittergent 3-12 and 4 M guanidine HCl, and the extracted nonradiolabeled proteoglycans were purified by density gradient centrifugation. As assessed by quantification of the unsaturated disaccharides released from the proteoglycans by chondroitinase ABC treatment, 10(6) rat serosal mast cells contained 2.4-4.5 micrograms of chondroitin sulfate proteoglycans. Analysis of the chondroitinase ABC digests by high performance liquid chromatography revealed the unsaturated disaccharides delta Di-4S, delta Di-diSB, and delta Di-diSE which were derived from GlcA----GalNAc-4-SO4, iduronic acid-2-SO4----GalNAc-4-SO4, and GlcA----GalNAc-4,6-diSO4, respectively. The molar ratio of the monosulfated to disulfated disaccharides was approximately 2:1 with delta Di-diSE greater than delta Di-diSB. When analyzed with a mouse anti-chondroitin sulfate monoclonal antibody and fluorescein-labeled F(ab')2 goat anti-mouse IgG, approximately 91% of permeabilized and chondroitinase ABC-treated cells in the mast cell preparations exhibited intracellular fluorescence, and the pattern of staining indicated that the chondroitin sulfate molecules were located in the secretory granules. The specificity of the monoclonal antibody for the unsaturated double bond created by chondroitinase ABC treatment of the proteoglycan in situ was established by the absence of fluorescence when the chondroitinase ABC step was omitted or when heparinase digestion was substituted for chondroitinase ABC. Furthermore, the ability of the anti-chondroitin sulfate monoclonal antibody to mediate fluorescence in situ was markedly reduced by absorption with solid-phase chondroitin sulfate proteoglycan that had been chondroitinase ABC-treated, but not by absorption with undigested proteoglycan or with solid-phase heparin. The highly sulfated chondroitin sulfate proteoglycans of rat serosal mast cells are the same type synthesized by the rat mucosal mast cell subclass.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1967

Regulation of cardiac muscle contractility.

The heart's physiological performance, unlike that of skeletal muscle, is regulated primarily by variations in the contractile force developed by the individual myocardial fibers. In an attempt to identify the basis for the characteristic properties of myocardial contraction, the individual cardiac contractile proteins and their behavior in contractile models in vitro have been examined. The low shortening velocity of heart muscle appears to reflect the weak ATPase activity of cardiac myosin, but this enzymatic activity probably does not determine active state intensity. Quantification of the effects of Ca(++) upon cardiac actomyosin supports the view that myocardial contractility can be modified by changes in the amount of calcium released during excitation-contraction coupling. Exchange of intracellular K(+) with Na(+) derived from the extracellular space also could enhance myocardial contractility directly, as highly purified cardiac actomyosin is stimulated when K(+) is replaced by an equimolar amount of Na(+). On the other hand, cardiac glycosides and catecholamines, agents which greatly increase the contractility of the intact heart, were found to be without significant actions upon highly purified reconstituted cardiac actomyosin.

Adenosine Triphosphatases↗

PubMed1986

Estrogen receptor immunocytochemical assay (ER-ICA) in human endometrium.

An estrogen receptor immunocytochemical assay (ER-ICA) was applied to 15 tissue samples from human endometrium: five proliferative, five secretory, three carcinomas, and two atypical hyperplasias. A monoclonal anti-ER (H 222 SP gamma, Abbott Lab.) and peroxidase antiperoxidase method were applied on frozen sections, 5 micron thick for light microscopy (LM), 100 micron thick for electron microscopy (preembedding). Positive ER staining was quantitated on tissue sections (LM) using a computerized system of image analysis referred to as SAMBA 200 (Thomson TITN). Positive immunostaining was observed in the nuclei of both epithelial and stromal cells in normal and disordered endometrium. SAMBA 200 quantitative analysis permitted an accurate quantification of ER-positive staining. From this preliminary study it is concluded that (a) ER-ICA constitutes a reliable method to study the ER heterogeneous distribution in tissues and the precise intracellular ER localization, which is not feasible by ER biochemical binding assays; (b) SAMBA 200 analysis of the immunostained tissue sections permits an accurate and reproducible method of evaluating the results to quantitate the staining intensity and to determine the percentage of positive cells and the distribution of positive staining of the various tissue structures (glands and stroma).

Adenocarcinoma↗

PubMed1989

Epidermal growth factor induction of cellular proliferation and protooncogene expression in growth-arrested rat H4IIE hepatoma cells: role of cyclic adenosine monophosphate.

The precise molecular events involved in growth factor-mediated cell proliferation in eukaryotes have not been entirely elucidated. Identification and characterization of the itnracellular molecular signaling systems linking growth factor function with nuclear events would provide insight into the regulatory mechanisms governing eukaryotic cell growth. In this report, we demonstrate that serum-deprived rat H4IIE hepatoma cells enter a quiescent state and remain viable in the absence of serum for up to 7 days. These cells can be stimulated to transverse the cell cycle and proliferate in response to epidermal growth factor (EGF) after a 24-h lag phase. We were able to completely mimic the mitogenic effects of EGF with 8-p-chlorophenylthio-cAMP (8-CPT-cAMP) but only partially with N6-(Bu)2-cAMP. EGF and 8-CPT-cAMP together induce a synergistic increase in H4IIE hepatoma cell proliferation. The calcium ionophore A23187 and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate had little effect on H4IIE cell proliferation. EGF treatment led to a rapid and transient increase of intracellular cAMP concentration. Both 8-CPT-cAMP and EGF were also equally effective in causing a rapid and transient induction of c-fos and c-myc protooncogene mRNA levels when added to growth-arrested H4IIE cells while A23187, N-(Bu)2-cAMP, and 4 beta-phorbol 12-myristate 13-acetate were significantly less effective. Both EGF and 8-CPT-cAMP affect protooncogene expression in growth-arrested rat H4IIE hepatoma cells primarily at the transcriptional level. Localization and semi-quantification of nuclear pp55c-fos and 63 (kilodalton)-myc protooncoproteins by immunocolloidal gold electron microscopy revealed that EGF and/or 8-CPT-cAMP treatment of quiescent H4IIE hepatoma cells led to a marked and rapid nuclear accumulation of these proteins in discrete nuclear substructures. Cummulatively, these results suggest that cAMP participates in the intracellular signaling system mediating the mitogenic and protooncogene inducing effects of EGF on growth-arrested rat H4IIE hepatoma cells.

Animals↗

PubMed1989

Calmodulin quantification and immunolocalization in developing embryonic orofacial tissue.

Temporally and quantitatively coordinated synthesis of cyclic adenosine monophosphate appears to be critical for normal development of the mammalian secondary palate. Calmodulin has been implicated as being involved in mediating the activity of a number of fundamental calcium-regulated intracellular enzyme systems including phosphodiesterases, adenylate cyclase, and a variety of kinases, all of which may regulate or be regulated by intracellular cAMP. Calmodulin levels were thus quantified, and endogenous calmodulin was immunolocalized in developing palatal tissue in vivo and in embryonic palatal mesenchymal cells in vitro. Endogenous palatal calmodulin levels, determined by radioimmunoassay, showed little variation during the period of murine palatal ontogenesis and averaged 0.23 ng/micrograms protein. Murine palate mesenchymal cells in monolayer, either in logarithmic growth or at confluency, contained similar levels of calmodulin. In palate mesenchymal cells in primary culture, specific anti-calmodulin staining was confined to the cell cytoplasm and was concentrated in the perinuclear region. Since immunostaining for calmodulin appeared to be associated with discrete cytoplasmic filaments, distribution of actin and tubulin were investigated. Immunostaining for tubulin in these cells was also localized to the perinuclear region, while immunolocalization of actin showed staining patterns, reflective of stress fibers, which were quite different from those seen for calmodulin. Immunostaining was seen in vivo in all regions of the palatal epithelium with superficial peridermal cells staining most intensely. Specific immunostaining was also evident in palatal mesenchyme, where a pericellular distribution was seen. Staining patterns were similar throughout the period of palatal ontogeny. In addition, a sharply defined localization of calmodulin to cartilagenous extracellular matrix was noted. This study provides a useful initial approach toward understanding the role calmodulin may play in embryonic orofacial development.

Animals↗

PubMed1987

Intracellular phospholipase activity in rat heart: comparison between endogenous and exogenous substrates.

Two methods were used to estimate the intracellular phospholipase activity in rat heart: one using exogenous radioactive substrate dispersed as unilamellar vesicles; the other using endogenous membrane hydrolysis and subsequent phospholipid and lysophospholipid separation by high-performance liquid chromatography and quantification by phosphorus determination. We found that the endogenous method provided a higher hydrolysis rate than the exogenous method and that phosphatidyl ethanolamine was a better substrate than phosphatidyl choline.

Animals↗

PubMed1991

Quantification of Ca(2+)-ATPases in porcine duodenum. Effects of 1,25(OH)2D3 deficiency.

Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.

Adenosine Triphosphate↗

PubMed1987

Propidium iodide as a nuclear counterstain for immunofluorescence studies on cells in culture.

We describe a rapid procedure using propidium iodide (PI) as a nuclear counterstain in immunofluorescence studies where cell surface or intracellular antigens are localized with fluorescein-conjugated antisera. In fixed monolayer preparations, all cell nuclei fluoresce red and can be seen simultaneously with cellular antigens that fluoresce green. Counterstaining with PI therefore makes possible quantification of the proportion of cells present in culture that stain immunocytochemically for a specific antigen.

Cell Nucleus↗

PubMed1989

Inflammatory cell and immune function in Merino sheep with chronic dermatophilosis.

Components of inflammatory and immunological responses were compared in 17 Merino sheep with chronic dermatophilosis (Group 1) and 15 Merino sheep that had recovered from the disease (Group 2). The functions studied included: (i) total and differential white cell counts; (ii) phagocytic function and intracellular killing by neutrophils; (iii) humoral immune response to T-dependent and T-independent antigens and to Dermatophilus congolensis. (iv) lymphocyte blastogenic responses to phytohaemagglutinin; (v) bovine serum albumen and D. congolensis antigens; (vi) quantification of T-lymphocyte subsets in skin lesions resulting after re-infection with D. congolensis zoospores. After all lesions were treated and the sheep were shorn, both groups of sheep were re-infected with D. congolensis. Both groups had similar infection rate, severity of lesions and rate of resolution after re-infection. The Group 2 sheep had significantly higher primary and secondary antibody responses to killed Brucella abortus cells than Group 1 sheep, but Group 1 sheep had higher levels of specific D. congolensis antibody throughout the trial. Neutrophils from Group 1 sheep showed a higher phagocytic rate for D. congolensis zoospores than Group 2 sheep when the zoospores were opsonised by sera from the Group 1 sheep, but there was no difference in their ability to kill ingested zoospores. Although there were some differences between the groups in the proportion of lymphocytes in lesions that reacted with monoclonal antibodies to T4, T8 and T19-19 lymphocyte markers at various times after re-infection, the sheep in Group 2 consistently had higher levels of lymphocytes reacting to a monoclonal antibody for the T6 lymphocyte antigen in skin biopsies collected 9, 15 and 21 days post-inoculation (p.i.) than did sheep in Group 1. Group 2 sheep also had higher levels of epidermal cells with immunohistochemical properties of Langerhans cells at lesion sites 15 and 21 days p.i.

Actinomycetales Infections↗

PubMed1985

Flow cytometric immunofluorescence of rat anterior pituitary cells.

We have developed a flow cytometric immunofluorescence technique for the quantification of growth hormone (GH), prolactin (PRL), and luteinizing hormone (LH) producing cells. The procedure requires about 24 hours and can objectively count 50,000 cells in about 3 minutes. It is based on indirect-immunofluorescence (fluorescein) of intracellular hormone using an EPICS V cell sorter. The fluorescein distributions are gated on DNA content (propidium iodide) to eliminate counting cell clumps. Cells from the same suspensions were stained immunocytochemically and counted microscopically (1,000-2,000 cells/sample). Immunofluorescence and immunocytochemistry correlated to within a few percent for GH and PRL cells. Cell suspensions from adult males and females with or without castration and a diethylstilbestrol (DES)-induced primary pituitary tumor were used to test the method. A major finding of this study was the objective identification of two populations of PRL producing cells, i.e., lightly and intensely stained cells. On the other hand, the fluorescence distribution of PRL cells from DES-induced pituitary tumors did not fall into two distinct populations but, rather, represented a broad continuum. This method should prove useful in studying the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

Animals↗

PubMed1978

"Runde" virus: further characteristics and a method for purification.

Neither BHK 21/c13, BSC-1, Vero nor GMK cells were of use for quantification of "Runde" virus. The titres were low and difficult to reproduce. Infected newborn mouse brains gave considerably higher yields than any of the cell cultures. The growth curve in BHK 21/c13 cells showed a slow increase in both intracellular and extracellular virus until maximum titres of about 10(6) baby mouse LD50 were reached at 48 and 72 hours post-infection (p.i.). During the following 24 hours, the infectivity dropped by about 1 log10 unit and was then unaltered until 196 hours p.i. Infected BHK 21/c13 cells did not haemadsorb chicken erythrocytes, although the culture media contained haemagglutinins. Resistance to BUdR indicated an RNA genome. Concentrated and purified virus preparations were produced by polyethylene glycol 6000/NaCl "precipitation" and hydroxylapatite column chromatography. Treatment with a colloidal silica gel widened the spectrum of agglutinable erythrocyte species.

Animals↗

PubMed1986

The immune response of a marine teleost, Pseudopleuronectes americanus, (winter flounder) to the protozoan parasite Glugea stephani.

G. stephani is an intracellular cyst-forming microsporidan parasite that is found in the intestine of winter flounder (WF) Pseudopleuronectes americanus. No detectable humoral response was seen in parasitized fish or in fish injected with either spores or spore homogenate from this parasite. Quantification of total immunoglobulin (Ig) levels showed a decrease in Ig levels rather than enhancement, 21 days after intramuscular (IM) injections of spores (3 X 10(6)/ml). When a second injection of spores was administered on day 21 and tested 3 weeks later, a further decrease in total serum Ig's occurred. A decrease in total IgM levels also occurred in WF that were simultaneously injected with G. stephani and the antigens, horse red blood cells (HRBC) or formalin-killed Klebsiella pneumonia (KP). The total Ig levels of fish injected with an antigen plus spores was not as low as those injected with the parasite alone. The Ig levels, as well as antibody titers to HRBC and KP were however, lower when compared to fish injected only with the HRBC or bacteria. Disrupted spore homogenate injected into winter flounder, showed a less marked decrease in Ig levels when compared with whole spores. When a single IM injection of spores was given, followed by two weekly injections of indomethacin (a drug that inhibits prostaglandin activity), no decrease in Ig levels occurred and levels were comparable to control (saline injected) fish.

Animals↗

PubMed1988

Relationship of prostatic acid phosphatase localization in human prostate by a monoclonal antibody with the Gleason grading system.

Prostatic acid phosphatase (PAP) was localized in human prostate with a monoclonal antibody prepared against PAP isoenzyme II to determine patterns of its expression in normal, hyperplastic (BPH), and cancerous glands. The monoclonal antibody reacted with both isoenzymes II and IV in immunoblot studies. Formalin-fixed, paraffin-embedded tissue was used from patients who had not been treated with hormones or chemotherapy. In normal glands and BPH, there was marked variation in the intensity of PAP staining in morphologically otherwise similar epithelial cells. There was similar heterogeneity of staining in the adenocarcinomas. Rough quantification of the intensity patterns in the clinical groups indicated a slight shift to more intense staining in BPH and well-differentiated carcinomas but a progressive decline in the PAP staining in the moderately and poorly differentiated tumors. This decrease in intracellular staining with decreasing differentiation is not inconsistent with the clinical observation that serum levels of acid phosphatase generally increase with higher grade and disseminated tumors, since the enzyme is simply more accessible to the circulatory system in those cases. The same decrease may explain the few disseminated tumors that are not associated with elevated serum levels.

Acid Phosphatase↗

PubMed1985

Inhibition of lectin-induced lymphocyte activation by 2-cyclohexene-1-one: analysis of DNA synthesis in individual cells by BUdR quenching of Hoechst 33258.

A novel technique utilizing the quenching of fluorescence Hoechst 32258 by bromodeoxyuridine (BUdR) was used to investigate the effect of depressed glutathione (GSH) on the activation of human peripheral blood lymphocytes by phytohemagglutinin (PHA) or concanavalin A (con A). This technique allows the quantification of DNA synthesis in individual cells. Lymphocytes were purified by Ficoll-Hypaque density gradient centrifugation and treated with 5 X 10(-5) M to 1 X 10(-6) M 2-cyclohexene-1-one (2-CHX-1), a reagent which specifically depletes intracellular GSH, and/or interferes with GSH-protein interactions, and 25 micrograms/ml BUdR in the presence or absence of PHA or con A. At 72 h lymphocyte smears were stained with Hoechst 33258 and examined using a computer controlled microscope photometer. When DNA synthesis was assayed using BUdR quenching two populations of lymphocytes were noted; a population which incorporated little or no BUdR (unactivated) and a population which incorporated BUdR sufficient to quench 33258 fluorescence by approximately 35%. Cells treated with graded doses of 2-CHX-1 which reduced glutathione levels by 10-90%, showed a progressive loss of cells from the activated population and the appearance of these cells in the inactivated population. Statistical analysis of the frequency histograms demonstrated that there were no cells which incorporated an intermediate amount of BUdR. This data demonstrates that depressed intracellular GSH or inhibition of GSH-protein interactions inhibits an early step in the biochemical sequence of events leading to DNA synthesis but does not inhibit the DNA synthetic process per se.

Benzimidazoles↗

PubMed1988

Dependence of metabolic and structural heterogeneity of cholesterol ester-rich very low density lipoproteins on the duration of cholesterol feeding in rabbits.

Cholesterol ester-rich (CER) VLDL accumulate rapidly in the plasma of rabbits fed cholesterol-enriched diets. However, the major loci of enhanced synthesis of subfractions of CER-VLDL, their interaction with macrophages, and their relative contribution to atherogenesis have not yet been elucidated. To determine whether anabolism is hepatic or intestinal, subfractions of CER-VLDL were characterized at selected intervals from day 0 to 60 of cholesterol feeding. Rate zonal ultracentrifugation of CER-VLDL from rabbits fed cholesterol for 4 and 60 d demonstrated an early increase of the proportion of cholesterol carried in the intestinally-derived fraction (designated as Fx-I) of VLDL compared with that in hepatically-derived particles (Fx-H). Quantification by size exclusion HPLC also demonstrated that Fx-I was a prominent CER-VLDL component at day 4, while Fx-H particles became increasingly prominent with further cholesterol feeding. At both 4 and 60 d Fx-I stimulated cholesterol esterification and intracellular cholesterol content in macrophages more than the corresponding Fx-H did. In fact, Fx-H harvested at 4 d produced no cholesterol ester deposition. In contrast, Fx-H harvested at 60 d markedly stimulated cholesterol esterification and intracellular cholesterol content. Thus, both compositional and metabolic characteristics of CER-VLDL changed as a function of the duration cholesterol feeding.

Animals↗

PubMed1989

Radiochemical diagnosis of adrenal disease.

The adrenal gland consists of cortical and medullary components, both of which may be functionally imaged using radiopharmaceuticals designed to take advantage of the specialized physiology of each tissue. Radiolabeled cholesterol derivatives are taken up by normal and abnormal adrenal cortex and provide an in vitro map of adrenocortical function in Cushing's Syndrome, hyperaldosteronism, and hyperadrenogenism. Quantification of uptake shows strong correlation with biochemical parameters of adrenocortical hyperfunction. In the case of incidentally discovered euadrenal masses scintigraphy may help to distinguish between benign, nonfunctional cortical adenomas, and destructive, potentially malignant lesions. Radiolabeled metaiodobenzylguanidine (MIBG) is a tracer of type I uptake and intracellular storage capacity for catecholamines in sympathomedullary tissues and tumors derived from this system. This permits the location of pheochromocytomas of all types (benign and malignant, intra- and extra-adrenal, sporadic and familial) as well as neuroblastomas in all stages. Other neuroendocrine tumors with type I uptake capacity may also be imaged. In suitable cases preliminary data suggests the administration of large doses of MIBG may permit internal radiotherapy of otherwise untreatable neuroendocrine tumors.

Adrenal Gland Diseases↗