Effect Of Prolonged Incubation Research Articles

Abstract 3371: Periostin secreted by breast cancer cells inhibits macrophage phagocytosis and adhesion to fibronectin

Abstract Interactions between cancer cells and immune cells are critical to the development of breast cancer metastases. We have previously shown that breast cancer cells secrete periostin, an extracellular matrix protein implicated in tumor progression. Here we evaluated 1) the effect of macrophage secretions on 4T1 breast cancer cell production of periostin and 2) the effect of periostin on key macrophage functions. In vitro, periostin secretion by highly metastastic 4T1 mammary cancer cells was investigated by ELISA. Following a 48-hour incubation with conditioned media obtained from J774A-1 monocyte cells, 4T1 cells secreted significantly higher periostin concentrations (p<0.05). Next, the effect of prolonged incubation with periostin on proliferation, adhesion and phagocytosis of macrophages was investigated using mouse J774A-1 and RAW264.7 monocyte cells and mouse primary bone marrow macrophages. In the conditions tested, periostin did not affect macrophage proliferation. Incubation with periostin significantly decreased macrophage adhesion to fibronectin-coated vessels (p<0.05). Incubation with periostin also significantly inhibited macrophage phagocytosis of polymer beads (p<0.05). Together, these observations indicate that the secretion of periostin by 4T1 cells is, in part, stimulated through paracrine communication with macrophages, and that periostin inhibits both macrophage adhesion and phagocytosis. Citation Format: Michelle M. Coleman, Rachel S. Helms, Didier Dreau. Periostin secreted by breast cancer cells inhibits macrophage phagocytosis and adhesion to fibronectin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3371. doi:10.1158/1538-7445.AM2015-3371

Effect of Time and Temperature on PrPCWD Immunoreactivity as Evidenced by Western Blot

The protease-resistant infectious prion protein, PrPres, that causes transmissible spongiform encephalopathies, is remarkably resistant to conventional physical and chemical sterilization methods, including heat. It was hypothesized that thermal-dependent PrPres degradation has been underestimated, and the effect of prolonged incubation at 37 degrees C, 55 degrees C, and 80 degrees C on PrPres detection was examined using brain homogenates from chronic wasting disease-affected elk and mule deer (PrPCWD). Immunoblotting demonstrated progressive loss of PrPCWD immunoreactivity with time in all incubated samples as temperature increased, and PrPCWD was virtually undetectable after 90 days of incubation at 55 degrees C and 80 degrees C. These results indicate that decontamination methods and tissue disposal systems maintaining elevated temperatures for long periods of time could interfere with immunodetection, and the reliability of assays for PrPres detection could be compromised when applied to tissues exposed to heat with time. Although these results may suggest that such prolonged heat treatment could destroy prions, the observed loss of immunoreactivity does not necessarily correlate with a concurrent loss of infectivity. Bioassay is needed to determine if samples that have been incubated under these conditions retain infectivity.

Effect of prolonged incubation with copper on endothelium-dependent relaxation in rat isolated aorta.

1 We investigated the effects of prolonged exposure to copper (Cu(2+)) on vascular functioning of isolated rat aorta. 2 Aortic rings were exposed to CuSO(4) (3-24 h) in Dulbecco's modified Eagle medium with or without 10% foetal bovine serum (FBS) and then challenged with vasoconstrictors or vasodilators in the absence of Cu(2+). 3 Exposure to 2 micro M Cu(2+) in the absence of FBS did not modify the response to phenylephrine (PE) or acetylcholine (ACh) in aortic rings incubated for 24 h. Identical exposure in the presence of FBS increased the contractile response to 1 micro M PE by 30% (P<0.05) and impaired the relaxant response to 3 micro M ACh or 1 micro M A23187 (ACh, from 65.7+/-7.1 to 6.2+/-1.1%, n=8; A23187, from 74.6+/-8.2 to 12.0+/-0.8%, n=6; P<0.01 for both). Cu(2+) exposure did not affect the relaxant response to NO-donors. 4 Impairment of vasorelaxation appeared 3 h after incubation with 2 micro M Cu(2+) and required 12 h to attain a steady state. Vasorelaxation to ACh was partially restored by 1 mM tiron (intracellular scavenger of superoxide ions; maximum relaxation 34.2+/-6.4%, n=10, P<0.01 vs Cu(2+) alone), whereas catalase, superoxide dismutase or cycloheximide were ineffective. 5 Twenty-four hour-exposure to 2 micro M Cu(2+) did not affect endothelium integrity or eNOS expression, and increased the Cu content in arterial rings from 6.8+/-1.1 to 18.9+/-2.9 ng mg(-1) wet weight, n=8; P<0.01. 6 Our results show that, in the presence of FBS, prolonged exposure to submicromolar concentrations of Cu(2+) impaired endothelium-dependent vasorelaxation in aortic rings, probably through an intracellular generation of superoxide ions. British Journal of Pharmacology (2002) 136, 1185-1193

Plant cells show an acquired insensitivity to Brefeldin A

The parameters of Brefeldin A (BFA) action in plant cells have been further investigated in maize root tips. Incubation of maize root tip cells with 100 µg ml−1 BFA for 2 h induces the vesiculation and aggregation of Golgi membrane to form BFA compartments; an effect that is reversed by incubation of the cells in BFA-free medium. In this paper, the effects of prolonged incubation with BFA are reported, revealing that Golgi stacks spontaneously re-form in the presence of this drug after incubation for 12 h or 24 h. The observation that the same BFA solution is still able to induce the forma- tion of BFA compartments in fresh roots suggests that this recovery is not due to metabolism, detoxification or depletion of the drug. Furthermore, incubation of maize roots with a fresh 100 µg ml−1 BFA solution for 2 h following 24 h BFA treatment fails to induce the reformation of BFA compartments. Thus, an acquired insensitivity to BFA in plant cells, that has not been observed in mammalian cells to date, is indicated.

Characterization of Radioiodinated Lung Surfactant Protein a (SP-A) and the Effects of Oxidation on SP-A Quaternary Structure and Activity

Lung surfactant protein A (SP-A) is the most abundant surfactant-associated protein present in the lung and respiratory tract. SP-A binds to several pathogens via its C-type lectin domains, and may act as an opsonin, mediating adhesion to cells via the collectin receptor. Binding studies using SP-A are made difficult by its apparent instability following radioiodination. This study investigated the effect of oxidation (via radioiodination and exposure to H2O2) on the structural and functional characteristics of SP-A. Radioiodinated SP-A, stored at 4 degrees C, retained carbohydrate binding activity after labeling. After 10 days storage, the radioiodinated SP-A was indistinguishable on SDS-PAGE from freshly radioiodinated SP-A, but sedimentation coefficient and Stokes radius values changed dramatically, indicating SP-A depolymerization. Such a quaternary structural breakdown, with a concomitant reduction in carbohydrate binding activity, is likely to be due to oxidative cleavage of disulfide bonds. Comparable results were observed upon radioiodination of the structurally similar molecule C1q. Consequently, the effect of prolonged incubation with H2O2 upon SP-A was investigated, with similar results. Thus, exposure to oxidizing agents leads to breakdown of the hexameric quaternary structure of SP-A, often to native polypeptides, with an attendant loss of binding activity. Such an effect may have consequences for the physiological role of SP-A in the lung.

Induction of Adhesiveness in Human Endothelial Cells by Plasmodium falciparum-Infected Erythrocytes

Cytoadhesion of infected erythrocytes to endothelium plays an important role in the pathogenesis of Plasmodium falciparum malaria. In vitro assays of cytoadhesion have helped to identify putative host ligands, namely thrombospondin, platelet glycoprotein IV (CD36), and intercellular adhesion molecule-1 (CD54) as possible mediators of cytoadhesion. However, the presence of these ligands on some host cells to which infected erythrocytes do not adhere raises the possibility that other molecules or factors may be involved. In the present study, we investigated the effects of prolonged incubation of endothelial cells (EC) with infected erythrocytes on adhesiveness of EC. We also studied the effects of tumor necrosis factor (TNF), interleukin-1 (IL-1), and phorbol myristate acetate (PMA). We found that when EC were incubated in contact with ring-infected erythrocytes for 24 hr during which the rings developed into trophozoites, adhesiveness was enhanced up to 250%. Incubation of EC with IL-1 or TNF for 12 hr increased adhesiveness by 50% at minimum doses of 5 U/ml and 50 U/ml, respectively, while PMA decreased adhesiveness in a consistent and dose-dependent manner. These results show that host EC adhesive ligands for infected erythrocytes can be induced, most notably by direct contact between the EC and infected erythrocytes containing developing parasites. The cultured human EC used in this study lacked surface CD36 detectable by immunofluorescence assay, suggesting that CD36 is not required for endothelial adhesiveness.

Glycogen content and glucose uptake in stimulated frog sartorius muscle: effects of prolonged incubation, insulin, and testosterone.

Anin vitro preparation of electrically stimulated sartorius muscle of the frog was used as a model to investigate the effects of insulin and testosterone on the increased glycogen content of frog skeletal muscle that was observedin vivo 48 h after an exhaustive bout of treadmill exercise. Thein vitro preparation could effectively extract glucose from the bathing medium and continue glycogen synthesis for incubation periods up to 60 h, and the electrical stimulation depleted glycogen by 68%. Electrical stimulation enhanced rates of glucose uptake and glycogen synthesis when these variables were measured after prolonged periods of poststimulation incubation but did not cause an overshoot of glycogen storage similar to that observedin vivo. Stimulation did not produce increments in glycogen synthesis that were additive to those of insulin or testosterone. The absence of glycogen overshoot in thein vitro system may have resulted from the absence of neural factors.

Effects of prolonged incubation and cell concentration on lipogenesis from glucose in isolated human omental fat cells

The behavior of human omental fat cells in vitro has been examined in order to define conditions under which glucose is converted to glyceride-glycerol and glyceride fatty acids. Synthesis of glyceride fatty acids from glucose reached maximal rates only after several hours of incubation in Krebs-Henseleit bicarbonate buffer, with or without added bovine albumin. Conversion of glucose to glyceride fatty acids was readily demonstrable with concentrated cell suspensions and was stimulated 3- to 8-fold by insulin. With dilute cell suspensions, little fatty acid was synthesized even after prolonged incubation in the presence of insulin. Conversion of glucose to glyceride-glycerol was linear during 6-hr incubations in buffer and unaffected by the concentration of the cell suspension. In the presence of bovine albumin, glyceride-glycerol synthesis was readily demonstrable at all cell concentrations used, although synthesis was faster in dilute suspensions. Thus, different incubation conditions produce widely divergent patterns of glucose metabolism in human omental fat cells.

The effect of prolonged incubation on RNA polymerase activity from surviving rat uteri

An 8-hr incubation of surviving immature rat uteri at 37 ° in tissue culture medium resulted in a 200% increase in DNA-dependent RNA polymerase activity (Nucleoside triphosphate: RNA nucleotidyl transferase, E.C. 2.7.7.6.). This increase in enzyme activity, shown previously to be dependent upon protein synthesis, has now been shown to require a temperature-sensitive (23 °) process as well. Since estradiol-17β-stimulated (4-hr) rat uterine DNA-dependent RNA polymerase activity is also controlled by protein and a similar temperature-sensitive process, the data suggest that the prolonged incubation of non-hormone-treated, immature rat uteri mimics the effect of estrogen.

The Effect of Prolonged Incubation on Lipid Synthesis by Rat Adipose Tissue

Abstract Prolonged incubation (6 hours) of adipose tissue from fed and 48-hour-fasted rats resulted in an increased rate of synthesis of fatty acids and glyceride glycerol from glucose uniformly labeled with 14C. However, lipogenesis from acetate-1-14C was increased by prolonged incubation in tissues from fasted animals only. These changes in the rate of lipid synthesis were not attributable to alterations in the content of free fatty acid in the tissue during incubation. The effect of prolonged incubation was demonstrable only in the absence of insulin or when very low concentrations were added. This was masked in the presence of a high concentration of insulin. Prolonged incubation increased the rate of glucose entry, as indicated by the more rapid accumulation of the nonmetabolizable sugar 3-O-methylglucose. It is concluded that the increase in utilization of glucose for lipid synthesis following prolonged incubation can be accounted for by the increased rate of glucose entry into the cells.

Effect of prolonged incubation on the electrophoretic pattern of amylase

Effect of prolonged incubation on the electrophoretic pattern of amylase

The addition of para-aminobenzoic acid or catalase to Löwenstein-Jensen medium and the effect of prolonged incubation in the culture of tubercle bacilli

A total of 1792 sputa from tuberculous patients before treatment of during treatment with isoniazid and PAS as cultured on medium with and without the addition of 10μg/ml. p -aminobenzoic acid. Of these, 528 specimens yielded positive cultures, the amount of growth and the speed of growth being the same on the two media. The failure to achieve improved results with the PABA medium has been attributed to dilution of PAS in the sputum by washing of the centrifuged deposit during culture. A total of 2814 sputa from patients mainly recciving isoniazid and PAS, isoniazid alone or no treatment, as cultured on Löwenstein-Jensen medium with and without the addition of 2μg. catalase. No difference was found between the media in the amount and the speed of growth among the 818 specimens yielding positive cultures, nor was the growth of isoniazid-resistant, catalase-negative organisms improved by the addition of catalase. In a sample of 1138 sputa, prolonging the period of incubation from 8–9 weeks to 16–17 weeks increased the percentage of positive cultures from 39.1% by only an additional 0.4%. The culture method emploted in controlled chemotherapeutic trials at the Tuberculosis Chemotherapy Centre, Madras, and in East Africa does not appear to have biased the bacteriological assessments of the results.

Action of muscular work on transfer of sugars across cell barriers; comparison with action of insulin.

Action of muscular work on transfer of sugars across cell barriers; comparison with action of insulin.