Central hemodynamics in rats with various resistivity to acute hemorrhage

OBJECTIVE AND DESIGN: Effects of hyperimmune milk factor (HIMF), an anti-inflammatory factor from milk of hyperimmunised cows, on tight junction permeability and cell growth were studied in vitro. MATERIAL OR SUBJECTS: Mammary (HC11) and kidney (MDCK) epithelial cell lines were used. TREATMENT: HIMF was used at a final concentration of 2 mg/ml. METHODS: Tight junction permeability was assessed by measuring transepithelial electrical resistance (TER) across confluent monolayers, following the addition of HIMF with or without an inflammatory challenge. Cell growth was assessed by measuring total DNA of cultures with and without HIMF. Data were analysed by analyses of variance. RESULTS: HIMF promoted tight junction formation and prevented loss of TER following a challenge in both epithelia. Post-challenge recovery of TER was also faster with HIMF. HIMF inhibited cell growth. CONCLUSIONS: HIMF stimulates tight junction maintenance and formation, and its previously reported anti-inflammatory properties may be mediated by restricting the extravasation of white blood cells through tight junctions.     

Partially purified soy hydrolysates retard proliferation and inhibit bacterial translocation in cultured C2BBe cells

Hydrolyzed soybean isolates SP-A and SP-B (Abbott Laboratories, OH), developed for use in enteral nutritional products, were tested in cultures of C2BBe cells, a colonic adenocarcinoma cell line with enterocytic differentiation, to evaluate effects on cell growth, maturation and ability to resist infection by enteric bacteria. SP-A delayed formation of confluent monolayers by 10 d compared with cells cultured without SP-A. SP-A also caused a retardation in the development of intercellular tight junctions as measured by transmonolayer electrical resistance (TER). SP-B had no effect on cell proliferation or TER of intestinal cell cultures. SP-A and SP-B enhanced the development of the brush border enzymes alkaline phosphatase and isomaltase over a 28 d period. By these criteria, SP-A and SP-B appear to affect intestinal epithelial cell development in culture. When C2BBe monolayers were exposed to the enteric bacteria, Salmonella typhimurium or Salmonella typhi, an inhibition of the passage of S. typhi was seen in cultures with SP-A and SP-B. No effect on the passage of S. typhimurium was seen with either soy isolate. Partially purified soy isolates therefore impart resistance to selected enteroinvasive bacteria. Addition of soy hydrolysates to the media of cultured intestinal cells may serve as a rapid and economical screening mechanism for preclinical trials that would test the therapeutic benefits of soybean isolates.     

Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-alpha

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp-like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.     

Dexamethasone induces posttranslational degradation of GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with the effects of fatty acids

GLUT2 expression is strongly decreased in glucose-unresponsive pancreatic beta cells of diabetic rodents. This decreased expression is due to circulating factors distinct from insulin or glucose. Here we evaluated the effect of palmitic acid and the synthetic glucocorticoid dexamethasone on GLUT2 expression by in vitro cultured rat pancreatic islets. Palmitic acid induced a 40% decrease in GLUT2 mRNA levels with, however, no consistent effect on protein expression. Dexamethasone, in contrast, had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about 65%. The effect of dexamethasone was more pronounced at high glucose concentrations and was inhibited by the glucocorticoid antagonist RU-486. Biosynthetic labeling experiments revealed that GLUT2 translation rate was only minimally affected by dexamethasone, but that its half-life was decreased by 50%, indicating that glucocorticoids activated a posttranslational degradation mechanism. This degradation mechanism was not affecting all membrane proteins, since the alpha subunit of the Na+/K+-ATPase was unaffected. Glucose-induced insulin secretion was strongly decreased by treatment with palmitic acid and/or dexamethasone. The insulin content was decreased ( approximately 55 percent) in the presence of palmitic acid, but increased ( approximately 180%) in the presence of dexamethasone. We conclude that a combination of elevated fatty acids and glucocorticoids can induce two common features observed in diabetic beta cells, decreased GLUT2 expression, and loss of glucose-induced insulin secretion.     

Dexamethasone enhances colonization of soft agar by tumorigenic mouse lung-derived cell lines

When cultured on plastic, tumorigenic mouse lung-derived cell lines exhibit different proliferative responses to glucocorticoids; some lines are inhibited while others are stimulated or unaffected. In contrast to the variable dexamethasone responses when cells are cultured on plastic, soft agar colonization by each of these cell lines is enhanced by dexamethasone. Enhanced soft agar growth is unlikely to result from expression of a mutant glucocorticoid receptor, since dexamethasone also enhanced colony formation in two cell lines that stably express a transfected normal glucocorticoid receptor gene. Thus, cell attachment influences the effect of glucocorticoids on cell cycle progression.     

Glucocorticoids stimulate growth of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes and support HPV16-mediated immortalization without affecting the levels of HPV16 E6/E7 mRNA

We investigated the effects of the glucocorticoids hydrocortisone and dexamethasone on human papillomavirus type 16 (HPV16)-mediated human cell carcinogenesis using normal human keratinocytes (HKc) and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16). Normal HKc did not require glucocorticoids for proliferation. In contrast, growth of early passage HKc/HPV16 strictly required these hormones, although glucocorticoid dependence became less stringent during in vitro progression. Glucocorticoid dependence was acquired by HKc early after immortalization with HPV16 DNA, and glucocorticoids were required for efficient HKc immortalization. However, treatment of HKc/HPV16 with hydrocortisone or dexamethasone did not increase the steady-state levels of HPV16 E6/E7 mRNA or protein. Firefly luciferase activity expressed under the control of the HPV16 upstream regulatory region and P97 promoter increased by about fourfold following dexamethasone treatment of HeLa, but only twofold in HKc/HPV16, and less than twofold in SiHa. However, all of these cell lines expressed sufficient endogenous glucocorticoid receptors to allow for a dexamethasone response of the mouse mammary tumor virus promoter. These results indicate that mechanisms other than a direct influence by glucocorticoids on HPV16 early gene expression may contribute to the striking biological effects of these steroids on HPV16-mediated human cell carcinogenesis.     

Glucocorticoid binding in mammary tissue slices of cattle in various reproductive states

Unlabeled cortisol and dexamethasone reduced tritiated cortisol and tritiated dexamethasone binding to 700 X g supernatant and precipitate fractions of mammary tissue slices from virgin heifers and from multiparous cows that were 1-mo prepartum (nonlactating), lactating (non-pregnant), or dry (nonpregnant, nonlactating). Unlabeled progesterone, testosterone, and 17beta-estradiol had no effect on tritiated glucocorticoid binding in 700 X g supernatant and precipitate fractions from these mammary tissue slices. The 700 X g fractions in mammary tissue slices from all cattle bound cortisol and dexamethasone with high affinity (Kd 10-10M). There were 1263 and 1955 molecules of cortisol and dexamethasone bound per mammary cell, respectively, in mammary tissue slices from lactating non-pregnant cows; in comparison virgin heifers bound 413 and 651 molecules of cortisol and dexamethasone; dry, non-pregnant cows bound 336 and 536 molecules of cortisol and dexamethasone; and 1-mo prepartum nonlactating cows bound 532 molecules of cortisol. Mammary tissue slices from cattle in reproductive states examined contained a major non-specific component which bound cortisol in both 700 X g tissue fractions. Since mammary tissue slices from lactating cattle bound more molecules of glucocorticoids than mammary slices from cattle in other reproductive states, we speculate specific glucorticoid binding may be associated with lactation.     

In-vitro cell culture models of the nasal epithelium: a comparative histochemical investigation of their suitability for drug transport studies

PURPOSE: To evaluate different in-vitro cell culture models for their suitability to study drug transport through cell monolayers. METHODS: Bovine turbinate cells (BT; ATCC CRL 1390), human nasal septum tumor cells (RPMI, 2650; ATCC CCL 30), and primary cell cultures of human nasal epithelium were characterized morphologically and histochemically by their lectin binding properties. The development of tight junctions in culture was monitored by actin staining and transepithelial electrical resistance measurements. RESULTS: The binding pattern of thin-sections of excised human nasal respiratory epithelium was characterized using a pannel of fluorescently-labelled lectins. Mucus in goblet cells was stained by PNA, WGA and SBA, demonstrating the presence of terminal N-acetylglucosamine, N-acetylgalactosamine and galactose residues respectively in the mucus of human nasal cells. Ciliated cells revealed binding sites for N-acetylglucosamine, stained by WGA, whereas Con A, characteristic for mannose moieties, labelled the apical cytoplasm of epithelial cells. Binding sites for DBA were not present in this tissue. Comparing three different cell culture models: BT, RPMI 2650, and human nasal cells in primary culture using three lectins (PNA, WGA, Con A) as well as intracellular actin staining and transepithelial electrical resistance measurements we found, that only human nasal epithelial cells in primary culture showed differentiated epithelial cells, ciliated nasal cells and mucus producing goblet cells, which developed confluent cell monolayers with tight junctions. CONCLUSIONS: Of the in-vitro cell culture models studied, only human nasal cells in primary culture appears to be suitable for drug transport studies.     

Autocrine regulation of protein secretion in mouse mammary epithelial cells

Milk secretion is under autocrine control by an inhibitory milk protein, named FIL (feedback inhibitor of lactation). Lactating mammary acini and epithelial cells cultured on reconstituted basement membrane (EHS matrix) with lactogenic hormones were used to study the characteristics of autocrine inhibition. FIL inhibited milk protein secretion in lactating acini, but not in epithelial cells on EHS matrix. The latter's insensitivity to FIL was due to formation of multicellular structures termed mammospheres, in which cell surrounded a central luminal space. Cell polarization, and the formation of tight intercellular junctions prevented FIL access to the apical cell surface, which faced the mammosphere lumina. When apical access was permitted either by incomplete mammosphere formation or EGTA treatment, FIL inhibited mammosphere protein secretion to the same extent as in lactating acini. The study shows that autocrine inhibition by FIL occurs specifically through interaction with the apical surface of the mammary epithelial cell, and suggests the presence of a FIL receptor on this, but not the basolateral cell membrane.     

Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the neuromuscular junction and other cholinergic synapses. Insight into the mechanisms controlling AChE expression in skeletal muscle is important for understanding formation, plasticity, and various dysfunctions of the neuromuscular junction. We have investigated the mechanisms responsible for the decreased AChE activity in the fast rat sternomastoideus muscle after chronic glucocorticoid treatment. Under such conditions fast skeletal muscles become atrophic and loose 30-40% of their AChE activity. In order to establish at which level synthesis of AChE is affected by glucocorticoids, we studied the effects of chronic dexamethasone treatment at both AChE mRNA and mature enzyme levels. Reduced rate of AChE recovery after subtotal irreversible AChE inhibition was observed during the first week of dexamethasone treatment, but not later. Statistical analyses of four independent northern blots revealed unchanged AChE mRNA levels. At the same time, we observed more than 60% decrease in the (G1+G2)/A12 ratio of molecular forms at the expense of G forms. It has been generally accepted that globular G1 and G2 molecular forms are synthesized in the rough endoplasmic reticulum as precursors of asymmetric (A) AChE forms, assembled in the Golgi apparatus. Reduced levels of G1 and G2 AChE forms, in combination with unchanged AChE mRNA, are therefore consistent with the reports demonstrating that glucocorticoids downregulate muscle protein synthesis at the translational level. Our findings support but not entirely prove the concept that impaired translation and/or posttranslational control are the primary cause of decreased AChE activity in the glucocorticoid-treated muscle.     

Immunopathology of uveitis

Two major types of plaque-bearing adhering junctions are commonly distinguished: the actin microfilament-anchoring adhaerens junctions (AJs) and the desmosomes anchoring intermediate-sized filaments (IFs). Both types of junction usually possess the common plaque protein, plakoglobin, whereas the other plaque proteins and the transmembrane cadherins are mutually exclusive. For example, AJs contain E-, N-, or P-cadherin in combination with alpha- and beta-catenin, vinculin and alpha-actinin, whereas in desmosomes, desmogleins and desmocollins are associated with desmoplakin and one or several of the plakophilins (PP1-3). Here we describe a novel type of adhering junction comprising proteins of both AJs and desmosomes and the tight junction (TJ) plaque protein, ZO-1, in a newly established, liver-derived tumorigenic rat cell line (RMEC-1). By immunofluorescence microscopy, cell-cell contacts are characterized by mostly continuous-appearing lines which are usually resolved by electron microscopy as extended arrays of closely spaced small plaque subunits. These plaque-covered regions are positive for plakoglobin, alpha- and beta-catenin, the arm-repeat protein p120, vinculin, desmoplakin and protein ZO-1. They are positive for E-cadherin in cultures early on in passaging, but tend to turn negative for all known cadherins in densely grown cultures. On immunoblotting SDS-PAGE-separated proteins from dense-grown cell monolayers, "pan-cadherin" antibodies have reacted with a band at approximately 140 kDa, identified as N-cadherin by peptide fingerprinting of the immunoprecipitated protein, which for reasons not yet clear is modified or masked in immunolocalization experiments. The exact histological derivation of RMEC-1 cells is not known. However, the observations of several endothelial markers and the fact that all cells are rich in IFs containing vimentin and/or desmin, while only subpopulations also reveal IFs containing CKs 8 and 18, is suggestive of a mesenchymal, probably endothelial origin. We discuss the molecular relationship of this novel type of extended junction with other types of adhering junctions.     

Structural determination of N-2''-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine from species of Aspergillus

The barrier function, surface biochemistry, and morphology of confluent monolayers of endothelial cells isolated from different segments of the bovine lung vasculature [microvessels (BLMVEC), vein (BPVEC) and artery (BPAEC)] were grown in culture and compared. A number of common cell surface proteins were identified along with two proteins of 46 and 48 kDa found exclusively on BPVEC. Lectin affinity chromatography revealed multiple glycosylation differences. The lectins, Arachis hypogaea (AHA) and Lycopersicum esculentum (LEA) agglutinins, interacted with several glycoproteins of BLMVEC but not of BPAEC. Bandeiraea simplicifolia (BS-1) and Caragana arborescens (CAA) agglutinins recognized several glycoproteins of BPVEC and BPAEC but not BLMVEC. Permeabilities were much lower for BLMVEC than BPAEC or BPVEC monolayers, with a range of about 16-fold less for sucrose to 2-fold less for albumin. Electron microscopy revealed that BLMVEC have a greater surface density of plasmalemmal vesicles (approximately 4-fold) and more extensively developed intercellular junctions with more focal membrane adhesion sites per junction (approximately 9-fold) than the other cells. We conclude that: i) BLMVEC monolayers form a much more restrictive barrier to molecular transport as a result of the tighter junctional formation; and ii) endothelial surface glycoproteins may be differentially glycosylated depending on their segmental location within the vasculature.     

Microsurgical management of craniopharyngiomas--outcomes in 56 patients

PURPOSE: The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H2-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H2-antagonists on the function of the intestinal tight junctions. METHODS: Effect of the H2-antagonists on the tight junctions has been investigated using the transepithelial electrical resistance (TEER) and the transport of mannitol across the Caco-2 cell monolayers. RESULTS: Four different H2-antagonists caused an increase in the TEER across the Caco-2 cell monolayers, accompanied by a decrease in the permeability for mannitol. The effect was concentration-dependent and saturable. Ranitidine and famotidine, caused a decrease in their own transport rate across the Caco-2 cells. Ranitidine competitively inhibited the increase in TEER caused by famotidine, whereas compounds which represent molecular fragments of ranitidine had no effect. The relative potency of the four H2-antagonists in causing an increase in the TEER correlated inversely with the oral bioavailability of these compounds in humans. CONCLUSIONS: We hypothesize that the H2-antagonists exert their effect on the tight junctions of Caco-2 cells by modulation of interactions among proteins associated with the tight junctional complex.