Cell culture models of fatty acid overload: Problems and solutions

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.     

β1 Integrins Do Not Have a Major Role in Keratinocyte Intercellular Adhesion

Integrins of the epidermis have been implicated both in intercellular adhesion and in cell-substratum adhesion. In the present study the role of α2β1 and α3β1 integrins has been evaluated further using human keratinocyte culture. α3β1 but not α2β1 strongly colocalizes with talin in adhesion plaques, consistent with a role for the former in adhesion to endogenous matrix. Upon elevation of the extracellular Ca²⁺ concentration from 30 μM to 1.0 mM, which is known to induce the organization of intercellular junctions, all three integrin subunits redistribute to concentrate along the cell-cell borders, but α3 redistributes more slowly. Blocking antibody to E-cadherin, which has previously been shown to delay the establishment of cell-cell adhesion upon Ca²⁺ elevation, delays the redistribution of α2β1 and α3β1 integrins. Elevation of the Ca²⁺ concentration also induces a rapid morphological change in the keratinocytes and organization of the culture into colonies with tight cell-cell connections. Blocking antibodies to β1 or to α3, but not to α2, delays this morphological change and the organization into colonies; however, the effect is much more pronounced in subconfluent cultures. These data are consistent with the hypothesis that anti-β1 or anti-α3 antibodies affect cell-cell interactions primarily through their previously described inhibition of motility. Stratification of the culture, which follows the formation of intercellular interactions, is normal in the presence of blocking antibody to β1 integrin. In summary, these data suggest that integrins do not play a major role in intercellular keratinocyte adhesion, but may appear to do so under certain conditions because of their involvement in motility.     

Significant induction of spermidine/spermine N1-acetyltransferase without cytotoxicity by the growth-supporting polyamine analogue 1,12-dimethylspermine

The superinduction of the polyamine catabolic enzyme spermidine/spermine N¹-acetyltransferase (SSAT) has been implicated in the cell type-specific cytotoxic activity of some polyamine analogues. We now report that one polyamine analogue, 1, 12-dimethylspermine (DMSpm), produces a large induction of SSAT with no significant effects on growth in the human large cell lung carcinoma line, NCI H157. This cell line has been demonstrated to respond to other analogues with SSAT superinduction and cell death. Treatment of the lung cancer cell line with DMSpm produces a rapid increase in SSAT activity and a near complete depletion of the natural polyamines. Additionally, DMSpm supports cell growth in cells which have been depleted of their natural polyamines by the ornithine decarboxylase inhibitor, 2-difluoromethylornithine. The current results suggest that significant induction of SSAT can occur in the absence of cytotoxicity when the inducing polyamine analogue can support growth and that increased SSAT activity alone is not sufficient for cytotoxicity to occur. © 1995 Wiley-Liss Inc.     

A polarized human endometrial cell line that binds and transports polymeric IgA

Summary We have demonstrated that a human endometrial cell line, HEC-1, maintains a high transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surfaces of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23α. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.Squamous epithelial cells typically contain two prominent types of cell–cell junctions: the adherens junction and the desmosome. The adherens junction is an intercellular adhesion complex that is composed of a transmembrane protein (a classical cadherin) and numerous cytoplasmic proteins (α-catenin, β-catenin and plakoglobin, vinculin and α-actinin; for reviews see Takeichi, 1990; Geiger and Ayalon, 1992). The cadherins are directly responsible for adhesive interactions via a Ca²⁺-dependent, homotypic mechanism, i.e., in the presence of sufficient Ca²⁺, cadherin on one cell binds to an identical molecule on an adjacent cell. The desmosome, also an intercellular adhesion complex, is composed of at least two different transmembrane proteins (desmoglein and desmocollin) as well as several cytoplasmic proteins, including desmoplakins and plakoglobin (Koch and Franke, 1994). The transmembrane components of the desmosome are members of the broadly defined cadherin family and also require Ca²⁺ for adhesive activity. However, decisive experimental evidence for homophilic or heterophilic interactions between desmosomal cadherins via their extracellular domains has not yet been presented (Koch and Franke, 1994; Kowalczyk et al., 1996). While members of the cadherin family constitute the transmembrane portion of both adherens junctions and desmosomes, the different classes of cadherins are linked to different cytoskeletal elements by the cytoplasmic components of each junction. Specifically, the classical cadherins are linked to actin filaments and the desmosomal cadherins to intermediate filaments.The organization of the proteins within the adherens junction is well understood (for reviews see Kemler, 1993; Cowin, 1994; Wheelock et al., 1996). Specifically, the intracellular domain of cadherin interacts directly with either plakoglobin or β-catenin, which in turns binds to α-catenin (Jou et al., 1995; Sacco et al., 1995). α-Catenin interacts with α-actinin and actin filaments, thereby linking the cadherin/ catenin complex to the cytoskeleton (Knudsen et al., 1995; Rimm et al., 1995). Cadherin/catenin complexes include either plakoglobin or β-catenin but not both (Näthke et al., 1994). The importance of the classical cadherins to the formation of adherens junctions and desmosomes has been demonstrated. Keratinocytes maintained in medium with low Ca²⁺ (i.e., 30 μM) grow as a monolayer and do not exhibit adherens junctions or desmosomes; however, elevation of Ca²⁺ concentration induces the rapid formation of adherens junctions followed by the formation of desmosomes (Hennings et al., 1980; Tsao et al., 1982; Boyce and Ham, 1983; Hennings and Holbrook, 1983; O''Keefe et al., 1987; Wheelock and Jensen, 1992; Hodivala and Watt, 1994; Lewis et al., 1994). Simultaneous blocking with functionperturbing antibodies against the two classical cadherins (E- and P-cadherin) found in keratinocytes inhibits not only Ca²⁺-induced adherens junction formation but also severely limits desmosome formation (Lewis et al., 1994; Jensen et al., 1996). Consistent with these findings, expression of a dominant-negative cadherin by keratinocytes results in decreased E-cadherin expression and delayed assembly of desmosomes (Fujimori and Takeicki, 1993; Amagai, et al., 1995). These data suggest some form of cross-talk between the proteins of the adherens junction and those of the desmosome. One candidate protein that might mediate such cross-talk is plakoglobin, since it is the only known common component of both junctions.Plakoglobin is found to be associated with the cytoplasmic domains of both the classical cadherins and the desmosomal cadherins. Despite the high degree of identity between plakoglobin and β-catenin (65% at the amino acid level; Fouquet et al., 1992), β-catenin only associates with the classical cadherins and not with the desmosomal cadherins. In the adherens junction, plakoglobin and β-catenin have at least one common function, i.e., the linking of cadherin to α-catenin and thus to actin. However, there is emerging evidence that other functions of these two proteins are not identical. For example, in a study by Navarro et al. (1993), E-cadherin transfected into a spindle cell carcinoma was shown to associate with α- and β-catenin, but not with the low levels of endogenous plakoglobin. The transfected cells did not revert to a more epithelial morphology in spite of the presence of functional E-cadherin, and the authors suggested that the lack of plakoglobin may have prevented such morphological reversion.In the present study, we have tested the hypothesis that plakoglobin, through its interaction with E- or P-cadherin, serves as a regulatory molecule for desmosome organization. Even though plakoglobin is not an essential structural component of the adherens junction (Sacco et al., 1995), our data indicate that plakoglobin can function as a regulator of desmosome formation only when it is associated with a classical cadherin. Thus, we propose that plakoglobin has at least two functions: (a) as a structural component of the adherens junction and the desmosome and (b) as a signaling molecule that regulates communication between the adherens junction and the desmosome.     

Oxidatively Induced Structural Alteration of Glutamine Synthetase Assessed by Analysis of Spin Label Incorporation Kinetics: Relevance to Alzheimer's Disease

Abstract: The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the second-order reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated β-amyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.     

Seven-Day Triple Therapy with Lansoprazole, Clarithromycin, and Metronidazole for the Cure of Helicobacter pylori Infection: A Short Report

Background To refine our understanding of anti- Helicobacter pylori treatment regimens further, we evaluated the efficacy and safety of lansoprazole given in combination with clarithromycin and metronidazole for 7 days in an open-label, multicenter study.
Materials and Methods. H. pylori -positive patients self-administered lansoprazole, 30 mg; clarithromycin, 500 mg; and metronidazole, 500 mg bid for 7 days. Patients were assessed at pretreatment, at which time the presence of H. pylori was documented by rapid urease test or histology and culture, following study drug administration (week 1) for a brief evaluation only, and at least 4 weeks posttreatment (week 5), including endoscopy with collection of biopsy specimens for culture and histology testing.
Results. Of the 60 patients enrolled in the study, 59 had confirmed H. pylori infection, and 51 were included in an intent-to-treat analysis of efficacy. Primary metronidazole and clarithromycin resistance were observed in 84% and 8% of study patients, respectively. One month after the end of therapy, H. pylori infection was cured in 40 of 51 patients (78%); 95% confidence interval, 65%–89%). The triple-therapy regimen was well-tolerated, with only 2 patients (4%) requiring premature withdrawal from the study due to treatment-related adverse events. Taste perversion (15.0%) and diarrhea (11.7%) were the most frequently reported adverse events possibly or probably related to study medication during the treatment period.
Conclusion. Despite a high prevalence of metronidazole resistance, a 1-week, triple-drug combination of lansoprazole, clarithromycin, and metronidazole is effective treatment for and well-tolerated by patients with H. pylori infection.     

The molecular basis of disease resistance in rice

The rice gene Xa21 conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo), was isolated using a map-based cloning strategy. Compared with previously cloned genes, the structure of Xa21 represents a novel class of plant disease R genes encoding a putative receptor kinase (RK). This article proposes a model for the mode of action of Xa21 and summarizes our current knowledge of the modular basis of resistance in rice to bacterial leaf blight and blast.     

Retention of soluble 99mTc-DTPA in the human lung: 24-h postdeposition

Foster, W. Michael, Pamela T. Stetkiewicz, and Arthur N. Freed. Retention of soluble^99mTc-DTPA in the human lung: 24-hpostdeposition. J. Appl. Physiol. 82(4): 1378-1382, 1997.Clearance of low-molecular-weightsolutes, e.g., radiolabeled chelate diethylenetriaminepentaacetate(DTPA), across epithelial surfaces of distal airways and the lungparenchyma is a broadly used technique to assess epithelial integrity.It has been generally assumed that clearance of solute follows a simplefirst-order process and that DTPA clearance through the respiratoryepithelium and into blood and lymphatic channels is complete within afew hours. Using -camera imaging and a radiolabeled aerosol of^99mTc-labeled DTPA, we observed ineight healthy subjects lung retention of radioisotope ~24 hpostdeposition of the ^99mTc-DTPA.Residual lung retention at the 24-h end point averaged 6.0 ± 1.8 (SD)% of the amount of radioisotope initially deposited in the lung.This suggests that for normal healthy subjects a small amount of the^99mTc radioisotope, either in adissociated or chelated form, is nonpermeable or slowly cleared fromrespiratory tisssues.