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Inflammatory responses have been shown to induce hyperglycemia, yet the underlying mechanism is still largely unclear. GLP-1 is an important intestinal hormone for regulating glucose homeostasis; however, few studies have investigated the influence of digestive tract Salmonella infection on enteroendocrine L cell secretions. In this study, we established a model of Salmonella -infected piglets by oral gavage in order to analyze the effects of Salmonella infection on enteroendocrine L cell function. Furthermore, in vitro lipopolysaccharide (LPS) was administered to STC-1 cells to clarify its direct effect on GLP-1 secretion. The results showed that significantly increased blood glucose in the group of Salmonella -infected piglets was observed, and Salmonella infection decreased blood GLP-1 content. Then, ileal epithelium damage was observed by histological detection, and this was further verified by TUNEL staining. We identified activation of TLR signaling demonstrating up-regulated expressions of TLR4 and nuclear factor-kappa B (NF-ΚB). Furthermore, it was shown that Salmonella induced pyroptosis of enteroendocrine L cells and enhanced the secretion of IL-1β through augmenting gene and protein expressions of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a carboxyl-terminal CARD (ASC), Caspase 1, and gasdermin D (GSDMD). Meanwhile, in vitro LPS treatment induced the pyroptosis of STC-1 cells and reduced the secretion of GLP-1. Altogether, the results demonstrated that Salmonella infection can reduce secretion of GLP-1 by inducing pyroptosis of intestinal L cells, which may eventually result in hyperglycemia. The results provided evidence for the cause of hyperglycemia induced by inflammation and shed new light on glucose homeostasis regulation.

Roux-en-Y gastric bypass (RYGB)-induced glycemic improvement is associated with increases in glucagon-like-peptide-1 (GLP-1) secreted from ileal L-cells. We analyzed changes in ileal bile acids and ileal microbial composition in diet-induced-obesity rats after RYGB or sham surgery to elucidate the early and late effects on L-cells and glucose homeostasis. In early cohorts, there were no significant changes in L-cell density, GLP-1 or glucose tolerance. In late cohorts, RYGB demonstrated less weight regain, improved glucose tolerance, increased L-cell density, and increased villi height. No difference in the expression of GLP-1 genes was observed. There were lower concentrations of ileal bile acids in the late RYGB cohort. Microbial analysis demonstrated decreased alpha diversity in early RYGB cohorts which normalized in the late group. The early RYGB cohorts had higher abundances of Escherichia-Shigella but lower abundances of Lactobacillus, Adlercreutzia, and Proteus while the late cohorts demonstrated higher abundances of Escherichia-Shigella and lower abundances of Lactobacillus. Shifts in Lactobacillus and Escherichia-Shigella correlated with decreases in multiple conjugated bile acids. In conclusion, RYGB caused a late and substantial increase in L-cell quantity with associated changes in bile acids which correlated to shifts in Escherichia-Shigella and Lactobacillus. This proliferation of L-cells contributed to improved glucose homeostasis., (© 2021. The Author(s).)

This morphological and immunohistochemical study demonstrates that tumors currently known as "middle ear adenomas" are truly well-differentiated epithelial neuroendocrine tumors (NETs) composed of cells comparable to normal intestinal L cells, and therefore, these tumors resemble hindgut NETs. These tumors show consistent expression of glucagon, pancreatic polypeptide, PYY, and the transcription factor SATB2, as well as generic neuroendocrine markers and keratins. The same L cell markers are expressed by cells within the normal middle ear epithelium. These markers define a valuable immunohistochemical profile that can be used for differential diagnosis of middle ear neoplasms, particularly in distinguishing epithelial NETs from paragangliomas. The discovery of neuroendocrine cells expressing the same markers in non-neoplastic middle ear mucosa opens new areas of investigation into the physiology of the normal middle ear and the pathophysiology of middle ear disorders., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The molecular sensors underlying nutrient-stimulated GLP-1 secretion are currently being investigated. Peripheral administration of melanocortin-4 receptor (MC4R) agonists have been reported to increase GLP-1 plasma concentrations in mice and humans but it is unknown whether this effect results from a direct effect on the GLP-1 secreting L-cells in the intestine, from other effects in the intestine or from extra-intestinal effects. We investigated L-cell expression of MC4R in mouse and human L-cells by reanalyzing publicly available RNA sequencing databases (mouse and human) and by RT-qPCR (mouse), and assessed whether administration of MC4R agonists to a physiologically relevant gut model, isolated perfused mouse and rat small intestine, would stimulate GLP-1 secretion or potentiate glucose-stimulated secretion. L-cell MC4R expression was low in mouse duodenum and hardly detectable in the ileum and MC4R expression was hardly detectable in human L-cells. In isolated perfused mouse and rat intestine, neither intra-luminal nor intra-arterial administration of NDP-alpha-MSH, a potent MC4R agonist, had any effect on GLP-1 secretion (P ≥0.98, n = 5-6) from the upper or lower-half of the small intestine in mice or in the lower half in rats. Furthermore, HS014-an often used MC4R antagonist, which we found to be a partial agonist-did not affect the glucose-induced GLP-1 response in the rat, P = 0.62, n = 6). Studies on transfected COS7-cells confirmed bioactivity of the used compounds and that concentrations employed were well within in the effective range. Our combined data therefore suggest that MC4R-activated GLP-1 secretion in rodents either exclusively occurs in the colon or involves extra-intestinal signaling., Competing Interests: RK is employed by Novo Nordisk (Denmark), but was exclusively employed at University of Copenhagen during the conception and design of experiments forming the basis of this paper. Novo Nordisk was not involved in the conception of study, design and execution of the experiments, interpretation of data or writing of the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kuhre, Modvig, Jepsen, Kizilkaya, Bæch-Laursen, Smith, Reimann, Gribble, Rosenkilde and Holst.)

Synthetic glucagon-like peptide-1 (GLP-1) analogues are effective anti-obesity and anti-diabetes drugs. The beneficial actions of GLP-1 go far beyond insulin secretion and appetite, and include cardiovascular benefits and possibly also beneficial effects in neurodegenerative diseases. Considerable reserves of GLP-1 are stored in intestinal endocrine cells that potentially might be mobilized by pharmacological means to improve the body's metabolic state. In recognition of this, the interest in understanding basic L-cell physiology and the mechanisms controlling GLP-1 secretion, has increased considerably. With a view to home in on what an L-cell is, we here present an overview of available data on L-cell development, L-cell peptide expression profiles, peptide production and secretory patterns of L-cells from different parts of the gut. We conclude that L-cells differ markedly depending on their anatomical location, and that the traditional definition of L-cells as a homogeneous population of cells that only produce GLP-1, GLP-2, glicentin and oxyntomodulin is no longer tenable. We suggest to sub-classify L-cells based on their differential peptide contents as well as their differential expression of nutrient sensors, which ultimately determine the secretory responses to different stimuli. A second purpose of this review is to describe and discuss the most frequently used experimental models for functional L-cell studies, highlighting their benefits and limitations. We conclude that no experimental model is perfect and that a comprehensive understanding must be built on results from a combination of models., Competing Interests: RK and NP are employed by Novo Nordisk. Novo Nordisk manage had no influence on the conception or content of this review. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kuhre, Deacon, Holst and Petersen.)

Aims/hypothesis: Insulin-like peptide-5 (INSL5) is found only in distal colonic L cells, which co-express glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). GLP-1 is a well-known insulin secretagogue, and GLP-1 and PYY are anorexigenic, whereas INSL5 is considered orexigenic. We aimed to clarify the metabolic impact of selective stimulation of distal colonic L cells in mice., Methods: Insl5 promoter-driven expression of Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) was employed to activate distal colonic L cells (L distalDq ). IPGTT and food intake were assessed with and without DREADD activation., Results: L distalDq cell stimulation with clozapine N-oxide (CNO; 0.3 mg/kg i.p.) increased plasma GLP-1 and PYY (2.67- and 3.31-fold, respectively); INSL5 was not measurable in plasma but was co-secreted with GLP-1 and PYY in vitro. IPGTT (2 g/kg body weight) revealed significantly improved glucose tolerance following CNO injection. CNO-treated mice also exhibited reduced food intake and body weight after 24 h, and increased defecation, the latter being sensitive to 5-hydroxytryptamine (5-HT) receptor 3 inhibition. Pre-treatment with a GLP1 receptor-blocking antibody neutralised the CNO-dependent improvement in glucose tolerance but did not affect the reduction in food intake, and an independent group of animals pair-fed to the CNO-treatment group demonstrated attenuated weight loss. Pre-treatment with JNJ-31020028, a neuropeptide Y receptor type 2 antagonist, abolished the CNO-dependent effect on food intake. Assessment of whole body physiology in metabolic cages revealed L distalDq cell stimulation increased energy expenditure and increased activity. Acute CNO-induced food intake and glucose homeostasis outcomes were maintained after 2 weeks on a high-fat diet., Conclusions/interpretation: This proof-of-concept study demonstrates that selective distal colonic L cell stimulation has beneficial metabolic outcomes. Graphical abstract.

Objects: Glucagon-like peptide-1 (GLP-1) is secreted from intestinal L cells, enhances glucose-stimulated insulin secretion, and protects pancreas beta cells. However, few studies have examined hypernutrition stress in L cells and its effects on their function. Here, we demonstrated that a high-fat diet reduced glucose-stimulated secretion of GLP-1 and induced expression of an endoplasmic reticulum (ER) stress markers in the intestine of a diet-induced obesity mouse model., Methods: To clarify whether ER stress in L cells caused the attenuation of GLP-1 secretion, we treated the mouse intestinal L cell line, GLUTag cells with palmitate or oleate., Results: Palmitate, but not oleate caused ER stress and decreased the protein levels of prohormone convertase 1/3 (PC1/3), an essential enzyme in GLP-1 production. The same phenomena were observed in GLUTag cells treated with in ER stress inducer, thapsigargin. Moreover, oleate improved palmitate-induced ER stress, reduced protein and activity levels of PC1/3, and attenuated GLP-1 secretion from GLUTag cells., Conclusions/interpretation: These results suggest that the intake of abundant saturated fatty acids induces ER stress in the intestine and decreases GLP-1 production., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mouse 3T3 feeder layer has been utilized for epidermal and corneal epithelial cell culture to promote tissue-like cell stratification. However, the molecular mechanism underlying epithelial-feeder layer interactions remains poorly understood. Here, the feeder layer activity of six different mouse cell lines was examined in terms of the colony-forming efficiency (CFE) of primary limbal epithelial cells, including corneal epithelial stem/progenitor cells. When epithelial cells and feeder layers were separated by culture inserts, the CFE was significantly lower than that of epithelial cells, which were cultured with feeder cells on the same dish surfaces, implying that direct contacts between these cells and/or pericellular extracellular matrix (ECM) deposition by feeder layers have an important role in feeder layer activity. With TaqMan polymerase chain reaction assay, the gene expression of 29 ECM molecules and 32 cadherin family genes was profiled in two highest and two lowest cell lines in the CFE for limbal and oral mucosal epithelial cells. A significant difference in the expression correlated with the CFE was observed in six ECM molecules and four kinds of cadherin family genes. In these results, type VI collagen was confirmed to be able to promote the colony formation of epithelial cells in vitro effectively.

The molecular mechanisms whereby CD28 alone or associated with TCR can regulate FOXP3 expression are not understood, although the importance of CD28 as a pivotal regulator of CD4(+) CD25(+) FOXP3(+) T cells is well recognized. We previously demonstrated that unique CD28-induced, NF-κB-dependent signals were sufficient to activate FOXP3 transcription in human CD4(+) CD25(-) T cells; however, the exact mechanisms are currently unknown. In this study, we have identified novel κB-binding sites on FOXP3 gene and demonstrated that CD28 signals mediated FOXP3 trans activation by nuclear translocation of RelA/NF-κB and not of c-Rel. The occupancy of FOXP3 κB-binding sites by RelA dimers that correlated with histone acetylation and recruitment of Pol II were required both to initiate FOXP3 transcription and to control the promoter occupancy by NFAT. Interestingly, knockdown of RelA in CD4(+) CD25(-) T cells stimulated through TCR and CD28 significantly affected FOXP3 expression, confirming that also the transcriptional activation of FOXP3 gene by TCR in the presence of CD28-costimulatory signals is RelA-dependent. In conclusion, these data suggest a new mechanism by which FOXP3 is activated and supports the critical role of CD28 in the regulation of peripheral tolerance., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. VSV infection of well-known cell lines pretreated with IFN-beta results in a 10(4)-fold reduction in the release of infectious particles, with a concomitant abrogation in viral transcript and/or protein levels. However, in cell lines of neuronal lineage only a threefold reduction in viral transcript and protein levels was observed, despite the same 10(4)-fold reduction in released infectious virions, suggesting an assembly defect. Examination of VSV matrix (M) protein ubiquitination yielded no differences between mock- and IFN-beta-treated neuronal cells. Further analysis of potential post-translational modification events, by scintillation and two-dimensional electrophoretic methods, revealed IFN-beta-induced alterations in M protein and phosphoprotein (P) phosphorylation. Hypophosphorylated P protein was demonstrated by reduced (32)P counts, normalized by (35)S-cysteine/methionine incorporation, and by a shift in isoelectric focusing. Hypophosphorylation of VSV P protein was found to occur in neuronal cell lysates, but not within budded virions from the same IFN-beta-treated cells. In contrast, hyperphosphorylation of VSV M protein was observed in both cell lysates and viral particles from IFN-beta-treated neuronal cells. Hyperphosphorylated M protein was demonstrated by increased (32)P counts relative to (35)S-cysteine/methionine normalization, and by altered isoelectric focusing in protein populations from cell and viral lysates. Hyperphosphorylated VSV M protein was found to inhibit its association with VSV nucleocapsid, suggesting a possible mechanism for type I IFN-mediated misassembly through disruption of the interactions between ribonucleoprotein cores, and hyperphosphorylated M protein bound to the plasma membrane inner leaflet.

The mannose 6-phosphate (Man-6-P) lysosomal targeting signal on acid hydrolases is synthesized by the sequential action of uridine 5'-diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) and GlcNAc-1-phosphodiester alpha-N-acetylglucosaminidase ("uncovering enzyme" or UCE). Mutations in the two genes that encode GlcNAc-1-phosphotransferase give rise to lysosomal storage diseases (mucolipidosis type II and III), whereas no pathological conditions have been associated with the loss of UCE activity. To analyze the consequences of UCE deficiency, the UCE gene was inactivated via insertional mutagenesis in mice. The UCE -/- mice were viable, grew normally and lacked detectable histologic abnormalities. However, the plasma levels of six acid hydrolases were elevated 1.6- to 5.4-fold over wild-type levels. These values underestimate the degree of hydrolase hypersecretion as these enzymes were rapidly cleared from the plasma by the mannose receptor. The secreted hydrolases contained GlcNAc-P-Man diesters, exhibited a decreased affinity for the cation-independent mannose 6-phosphate receptor and failed to bind to the cation-dependent mannose 6-phosphate receptor. These data demonstrate that UCE accounts for all the uncovering activity in the Golgi. We propose that in the absence of UCE, the weak binding of the acid hydrolases to the cation-independent mannose 6-phosphate receptor allows sufficient sorting to lysosomes to prevent the tissue abnormalities seen with GlcNAc-1-phosphotranferase deficiency.

GLUT1, the most ubiquitously expressed member of the GLUT family of glucose transporters, can be acutely activated by a variety of cell stresses. Methylene blue activates glucose transport activity of GLUT1 in L929 fibroblast cells presumably by a redox cycling of MB, which generates an oxidative stress. Data shown here reveal that methyl-beta-cyclodextrin (MCD) blocks both the staining of cells and activation of glucose uptake by directly binding to MB. MCD binding to MB was qualitatively demonstrated by a significantly slower dialysis rate of MB in the presence of MCD. Analysis of the complete spectra of aqueous MB solutions and MB plus MCD solutions by a factor analysis program called SIVVU indicated that these equilibria can be modeled by three species: MB monomer, MB dimer, and MCD-MB inclusion complex. The molar extinction coefficients for each species from 500 to 700nm were determined. The equilibrium association constant (K(a)) for MB dimer formation was measured at 5846+/-30M(-1) and the K(a) for formation of the MCD-MB complex was 310+/-10M(-1). MCD also dramatically enhances the destaining rate of MB-stained cells. The loss of MB from the cell is tightly correlated with the loss of activated glucose uptake. This suggests that the MB activation of glucose uptake is likely not caused by its redox cycling, but more likely the result of a specific interaction between MB and a protein directly involved in the activation of GLUT1.

Background: The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation., Results: We show here that Wnt proteins are rapidly endocytosed by a clathrin- and dynamin-mediated process. While endocytosis has traditionally been considered a principal mechanism for receptor down-regulation and termination of signaling pathways, we demonstrate that interfering with clathrin-mediated endocytosis actually blocks Wnt signaling at the level of beta-catenin accumulation and target gene expression., Conclusion: A necessary component of Wnt signaling occurs in a subcellular compartment distinct from the plasma membrane. Moreover, as internalized Wnts transit partially through the transferrin recycling pathway, it is possible that a "signaling endosome" serves as a nexus for activated Wnt pathway components.

Background: Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies., Results: Candidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 x 105 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular processes, including: IGF-II expression/signalling, vesicular transport/cytoskeletal trafficking and apoptosis. Notably, several of the genes have been directly implicated in the replication of reovirus and other viruses at different steps in the viral lifecycle., Conclusions: Tagged sequence mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus infection. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus lifecycle and may provide targets for novel anti-viral therapies.

Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus-cell interactions. The properties of uninfected and HSV-1-infected L fibroblasts and gro29 cells investigated by protein assay, immunoblot, titration assay, immunofluorescence light microscopy and immunogold cryosection electron microscopy are reported. The ultrastructure of both HSV-1-infected L and gro29 cells confirmed primary envelopment of virions at the nuclear membranes followed by maturing multiple de-envelopments and re-envelopments in the endoplasmic reticulum and in the Golgi complex. The gro29 cells presented changed cytoskeleton, abolished egress of virions, and were defective in the trafficking of glycoproteins, giving rise to accumulation of viral particles and glycoproteins in the endoplasmic reticulum and the Golgi complex. The results suggest that gro29 cells harbour a causal underlying defect of the cytoskeleton in addition to the HSV-1-induced cytoskeletal changes.

After incubation of L929 cells with [14C]serine and various effectors an inverse correlation between label in ceramide and phosphatidylserine (PS) was displayed. This surprising behavior of the two metabolites prompted us to check whether serine of PS could be a source for ceramide synthesis. We therefore incubated L929 cells for 30 min in serum-free medium with L-phosphatidyl-L-[3-14C]serine in the presence or in the absence of cycloserine, an established inhibitor of serine palmitoyltransferase. During this short period L-phosphatidyl-L-[3-14C]serine labeled ceramide and this label was suppressed by cycloserine. Then L929 cells were grown for 16-18 h in the presence of L-phosphatidyl-L-[3-14C]serine. After this period the label was seen in sphingomyelin. Labeling of ceramide and sphingomyelin by serine from PS provides evidence for a new metabolic relationship between glycerophospholipids and sphingolipids.

Background: Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway., Results: We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP., Conclusions: Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

The accumulation of gadolinium loaded as gadopentetic acid (Gd-DTPA) in chitosan nanoparticles (Gd-nanoCPs), which were designed for gadolinium neutron-capture therapy (Gd-NCT) for cancer, was evaluated in vitro in cultured cells. Using L929 fibroblast cells, the Gd accumulation for 12 h at 37 degrees C was investigated at Gd concentrations lower than 40 ppm. The accumulation leveled above 20 ppm and reached 18.0+/-2.7 (mean+/-S.D.) microg Gd/10(6) cells at 40 ppm. Furthermore, the corresponding accumulations in B16F10 melanoma cells and SCC-VII squamous cell carcinoma, which were used in the previous Gd-NCT trials in vivo, were 27.1+/-2.9 and 59.8+/-9.8 microg Gd/10(6) cells, respectively, hence explaining the superior growth-suppression in the in vivo trials using SCC-VII cells. The accumulation of Gd-nanoCPs in these cells was 100-200 times higher in comparison to dimeglumine gadopentetate aqueous solution (Magnevist), a magnetic resonance imaging contrast agent. The endocytic uptake of Gd-nanoCPs, strongly holding Gd-DTPA, was suggested from transmission electron microscopy and comparative studies at 4 degrees C and with the solution system. These findings indicated that Gd-nanoCPs had a high affinity to the cells, probably contributing to the long retention of Gd in tumor tissue and leading to the significant suppression of tumor growth in the in vivo studies that were previously reported.

We previously demonstrated the protective effect of the small heat-shock protein against oxidative damage induced by tumor necrosis factor alpha. Here we have extended our studies of the possible role of Hsp25 in ionizing radiation-induced damage. For these studies, we transfected murine fibroblast L929 cells with the Hsp25 gene and selected three stably transfected clones. Hsp25 overexpression conferred radioresistance as detected by clonogenic survival and induction of apoptosis. Interestingly, the Hsp25-transfected cells showed an increase in the level of the anti-apoptosis molecule Bcl2. We also observed alterations of cell growth in the Hsp25-transfected cells. The cell cycle time of Hsp25-transfected cells was 3-4 h slower than that of vector-transfected control cells. Flow cytometry analysis of synchronized cells at late G(1) phase by mimosine treatment also showed the growth delay in Hsp25-overexpressing cells. In addition, reduced cyclin D1, cyclin A and Cdc2 levels and increased levels of Cdkn1a (also known as p21(Waf)) were observed in Hsp25-transfected cells, which probably caused the reduction in cell growth. In addition, synchronization by mimosine treatment only partially altered radioresistance in the Hsp25-transfected cells. Taken together, these data suggest that Hsp25-induced radioresistance is associated with growth delay as well as induction of Bcl2.

A low cation conductance and a high anion conductance are characteristic of normal erythrocytes. In sickle cell anemia, the polymerization of hemoglobin S (HbS) under conditions of low oxygen tension is preceded by an increase in cation conductance. This increase in conductance is mediated in part through Ca(++)-activated K(+) channels. A net efflux of potassium chloride (KCl) leads to a decrease in intracellular volume, which in turn increases the rate of HbS polymerization. Treatments minimizing the passive transport of ions and solvent to prevent such volume depletion might include inhibitors targeting either the Ca(++)-activated K(+) channel or the anion conductance. NS1652 is an anion conductance inhibitor that has recently been developed. In vitro application of this compound lowers the net KCl loss from deoxygenated sickle cells from about 12 mmol/L cells/h to about 4 mmol/L cells/h, a value similar to that observed in oxygenated cells. Experiments performed in mice demonstrate that NS1652 is well tolerated and decreases red cell anion conductance in vivo. (Blood. 2000;95:1842-1848)

Groups of CBA mice immunosuppressed with anti-thymocyte serum (ATS) treatment were xeno-transplanted with either HeLa human cervical carcinoma cells or genetically modified cells expressing the human tumor necrosis factor-alpha (TNF) gene (All cells). Both cell lines were highly resistant to the cytotoxic effects of TNF. If 3 x 10(6) tumor cells were inoculated s.c. into female mice, HeLa cells grew progressively into large tumors and killed 74% of the recipients, while TNF-expressing All cells caused fatal tumor growth only in 22% of the mice. 3 x 10(6) or 1.5 x 10(7). All cells produced progressive tumor growth and lethality in all male recipients. In sera of all the A11-cell-transplanted mice, biologically active TNF was detected shortly (4.5 h) after tumor inoculation (6 39 U/ml), decreasing to below detection level in the circulation by day 3. In recipients of 15 million A11 cells, circulating TNF reappeared and reached high levels (12-1000 U/ml) 3 to 7 weeks later, when the animals bore large tumors (14-23 mm). Generally, such mice became cachectic, severely anemic, hypothermic, and soon died. On account of calcium mobilization from bones, their serum Ca levels were high. Electron microscopy revealed severe liver damage, but there were no signs of chronic arthritis. These results suggest that ATS-treated mice xenotransplanted with TNF-gene-transfected A11 human tumor cells provide a new model for studying the pathophysiological and anti-tumor effects of TNF.

Murine L cells showed markedly high lethal thermosensitivity. Survivals from fractionated heating at 44 degrees C with variety of interval time at 37 degrees C (44 degrees C for 10 min--variety of interval time at 37 degrees C-44 degrees C for 10 min) increased markedly in accordance with elongation of the internal time; i.e. survival fraction of 0.9% from 44 degrees C for 20 min alone without the interval time to those of 25% from the fractionated heating with interval time at 37 degrees C for 3-10 hrs. Incidence of apoptosis of the L cells from heating at 44 degrees C for 6.5 min (LD50) increased from 7% immediately after the heating to 30% 6-12 hrs after the post-incubation time at 37 degrees C. Accumulation of both hsp72 and p53 proteins markedly increased after a heating at 44 degrees C for 10 min alone in accordance with elongation of post-incubation time at 37 degrees C, representing a peak 6 hrs after the post incubation. Status of p53 gene in L cells were determined with Reverse Transcription-Polymerase Chain Reaction-Single Strand Conformational Polymorphism (RT-PCR-SSCP), i.e. wild type.

When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel filtration column chromatography. In this fraction, GSK-3beta, beta-catenin, and APC were co-precipitated with Axin. Although beta-catenin was detected in the high molecular weight fraction in L cells on gel filtration column chromatography, addition of conditioned medium expressing Wnt-3a to the cells increased beta-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of beta-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to beta-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3beta, beta-catenin, and APC, resulting in the stimulation of the degradation of beta-catenin and that Wnt-3a induces the dissociation of beta-catenin from the Axin complex and accumulates beta-catenin.

The function of the beta-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb beta-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a beta-globin locus (beta-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb beta-YAC that encompasses the entire beta-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). The Ppo-155 beta-YAC was used to directly lipofect MEL 585 cells. In 7 beta-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, epsilon + gamma + beta) per copy of integrated beta-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the beta-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked beta-YAC DNA. To test whether passage of the beta-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(beta-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human beta-globin mRNA (ie, epsilon + gamma + beta) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Expression of the T1 gene, also known as ST2, DER4, and Fit-1, has been shown to be associated with cell proliferation. It gives rise to two different mRNAs that encode a receptor-like protein and a soluble molecule representing the ectodomain of the receptor form. Although T1 is a member of the IL-1R family, its biologic function is currently unknown. In this study, we have analyzed the expression of the T1 surface Ag in murine hemopoietic organs. Mast cells (MCs) were shown to be the only identifiable cell lineage that expressed T1 at high levels. T1 expression was found on cultured bone marrow-derived immature MCs. Similarly, freshly isolated connective tissue-type MCs from the i.p. cavity were also shown to express high levels of T1. Interestingly, the earliest detectable committed MC precursor isolated from fetal blood (FB) at day 15.5 of gestation, but not circulating hemopoietic stem cells in FB, also expresses high level of T1. Since FB promastocytes lack expression of the high affinity IgE receptor (Fc epsilonRI), T1 expression precedes expression of Fc epsilonRI in MC ontogeny. The finding that the T1 Ag is selectively expressed at several stages during development of the MC lineage suggests that this cell surface molecule, in combination with the well-established markers c-Kit and Fc epsilonRI, should be valuable for studying the MC lineage.

Small stress proteins protect against the cytotoxicity mediated by oxidative stress. The relationship between Hsp25 expression and the integrity of the actin network was studied in H2O2-treated murine L929 fibrosarcoma cells overexpressing endogenous wild-type (wt-) or non-phosphorylatable mutant (mt-) Hsp25. We show here that both proteins prevented actin network disruption induced by a 1 h treatment with 400 microM H2O2. In contrast, SB203580, a p38 MAPkinase inhibitor which suppresses Hsp25 phosphorylation, abolished the protective activity conferred by both wt- and mt-Hsp25. Hence, phosphorylation does not appear essential for Hsp25 protective activity against H2O2-induced actin disruption, and SB203580-sensitive events other than Hsp25 phosphorylation may be important for actin network regulation. Since, in L929 cells, wt- or mt-Hsp25 expression modulates intracellular glutathione levels, analyses were performed which revealed a direct correlation between glutathione and the integrity of the actin network. Moreover, laser scanning confocal immunofluorescences revealed that only a small fraction of wt- or mt-Hsp25 colocalized with actin microfilaments. Taken together, our results suggest that, in L929 cells, the protection against actin network disruption is probably a consequence of the redox change mediated by Hsp25 rather than a direct effect of this stress protein towards actin.

Plasminogen-activator inhibitor type I (PAI-1), the primary inhibitor of urinary-type plasminogen activator, is thought to play an important role in the control of stroma invasion by both endothelial and tumor cells. Using an in vitro angiogenesis model of capillary extension through a preformed monolayer, in conjunction with in situ hybridization analysis, we showed that PAI-1 mRNA is specifically induced in cells juxtaposed next to elongating sprouts. To further establish that PAI-1 expression is induced as a consequence of a direct contact with endothelial cells, coculture experiments were performed. PAI-1 mRNA was induced exclusively in fibroblasts (L-cells) contacting endothelial cell (LE-II) colonies. Reporter gene constructs driven by a PAI-1 promoter and stably transfected into L-cells were used to establish that both mouse and rat PAI-1 promoters mediate apposition-dependent regulation. This mode of PAI-1 regulation is not mediated by plasmin, as an identical spatial pattern of expression was detected in cocultures treated with plasmin inhibitors. Because endothelial cells may establish direct contacts with fibroblasts only during angiogenesis, we propose that focal induction of PAI-1 at the site of heterotypic cell contacts provides a mechanism to negate excessive pericellular proteolysis associated with endothelial cell invasion., (Copyright 1998 by The American Society of Hematology.)

Sustained stimulation of the beta2-adrenoceptor leads to a desensitization of the receptor-mediated adenylyl cyclase stimulation. While desensitization promoted by nanomolar concentrations of isoproterenol involves the phosphorylation of the beta2-adrenoceptor by protein kinase A alone, both protein kinase A- and beta-adrenoceptor kinase-mediated phosphorylation leading to the binding of beta-arrestin contribute to the desensitization evoked by micromolar concentrations of agonist. In the present study, we assessed the influence of receptor density on the patterns of desensitization induced by these two different levels of stimulation. Murine L cells were transfected with a cDNA encoding the human beta2-adrenoceptor and clonal cell lines expressing various levels of beta2-adrenoceptor were used for the study. In cell lines expressing the highest number of receptor, approx. 150000 sites/cell (approx. 3000 fmol/mg of membrane proteins), pretreatment with micromolar concentrations of isoproterenol causes a desensitization pattern characterized by a reduction in both the potency and the efficacy of isoproterenol to further stimulate the adenylyl cyclase activity. In contrast, desensitization induced by 10 nM isoproterenol resulted only in a decrease in the potency of isoproterenol. This distinct pattern of desensitization is not seen in cells expressing 12000 receptors/cell (approx. 200 fmol/mg of membrane proteins) and, in that case, pretreatment with 10 nM isoproterenol leads to a reduction in both the sensitivity and the maximal response. Similar effects on the beta-adrenoceptor-stimulated adenylyl cyclase were observed in these cells following treatment with dibutyryl cAMP. Receptor density therefore dramatically influences the pattern of desensitization evoked by low level of stimulation. The results also demonstrate that although different molecular events are involved in the desensitization evoked by different levels of stimulation, its phenotypic expression can be qualitatively identical in cells expressing a relatively small number of receptors. Hence, protein kinase A-mediated desensitization cannot be qualitatively distinguished from the beta-adrenoceptor kinase-mediated process in these cells.

M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK- cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with alpha-catenin/beta-catenin or with alpha-catenin/plakoglobin, as E-and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence.

The relative function of the 15 and 13.2 kDa forms of SCP-2 in cholesterol trafficking and metabolism was assessed using L-cell fibroblasts permanently transfected with the cDNA encoding for either the mouse 15 kDa or 13.2 kDa SCP-2. Expression of the 15 kDa, but not the 13.2 kDa SCP-2 increased [3H]cholesteryl ester formation from medium derived cholesterol by 30% compared to control cells. In both SCP-2 expressing cell lines, sphingomyelinase treatment increased the initial rate of [3 H]cholesteryl ester formation from plasma membrane derived cholesterol more than 11-fold and elevated [3H]cholesteryl ester levels 1.5-fold compared to control cells. Expression of both proteins resulted in nearly a 1.5-fold increase in [3H]oleic acid esterification into cholesteryl esters, although [3H]oleic acid esterification into triacylglycerols was also increased in the 13.2 kDa SCP-2 expressing cells relative to control. In both transfected cell lines, the cholesteryl ester mass was increased nearly 2-fold compared to control cells, consistent with increased cholesteryl ester synthesis. Similarly, triacylglycerol levels were increased 1.3-fold in the 13.2 kDa SCP-2 expressing cells which is consistent with the increased [3H]oleic acid esterification into triacylglycerol. In the 15 kDa SCP-2 expressing cells, triacylglycerol levels were decreased 60%, but free cholesterol levels were increased 1.2-fold relative to control cells. Thus, only the 15 kDa expression product, containing the putative targeting sequence, specifically enhanced cholesteryl ester formation from either plasma membrane or medium-derived cholesterol. In contrast, the 13.2 kDa expression product, lacking the putative targeting sequence, stimulated an increase in [3H]oleic acid esterification into both cholesterol and triacylglycerol pools, suggesting a non-specific stimulation of fatty acid esterification.

The ability to maintain germinal center (GC) B cells in culture should facilitate studies on the molecular and cellular events which accompany affinity maturation and the generation of memory in T-dependent responses. We have investigated the ability of cytokines to maintain human tonsillar GC B cells (IgD-/CD39-/CD38+/CD77+) in the "CD40 culture system". In the absence of added cytokines, CD40 monoclonal antibody held on CD32-transfected L cells effectively sustained DNA synthesis in GC B cells for a maximum 3 to 4 days. Of the following cytokines (interleukin-1 beta [IL-1 beta], IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, and stem cell factor), only IL-2 and IL-4 provided a significant enhancement to DNA synthesis in the CD40 culture system; this was modest and short-term. Following a study on the cooperative activity between pairs of cytokines, triple combinations were identified that could maintain high levels of GC B-cell stimulation for at least 10 days. IL-10 was a common component of these synergistic cytokine cocktails, which were IL-10 + IL-4 + IL-7; IL-10 + IL-3 + IL-7; IL-10 + IL-1 beta + IL-2; IL-10 + IL-1 beta + IL-3, and IL-10 + IL-3 + IL-6. Culture of GC B cells with these cytokine combinations resulted in a net increase in viable cell numbers of 50% to 100% whereas total cell numbers increased up to fourfold. Cells recovered from these cultures retained a GC B-cell phenotype with a significant proportion being CD38+/CD44-, features characteristic of centroblasts. Studies with metabolically inactive CD32-L cells supported a role for stromal cell-derived soluble factors in maintaining GC B cells in vitro.

We examined the effects of insulin on fatty acid uptake in L-cell fibroblasts, using cis-parinaric acid to measure uptake rates in the absence of esterification and [3H]oleic acid to measure uptake rates in the presence of esterification. L-cells exhibited both high and low affinity insulin binding sites with Kd of 23 nM and 220 nM and a cellular density of 1.4 and 6.8 x 10(5) sites/cell, respectively. Insulin in the range 10(-9) to 10(-7) M significantly decreased both the initial rate and maximal extent of cis-parinaric acid uptake by 24 to 30%. Insulin also reduced [3H]oleic acid uptake up to 35%, depending on insulin concentration and decreased the amount of fatty acid esterified into the phospholipids and neutral lipids by 28 and 70%, respectively. In contrast, glucagon or epinephrine stimulated both the initial rate and extent of cis-parinaric acid uptake 18 and 25%, respectively. Because L-cells lack P-adrenergic receptors, the epinephrine effect was not the result of P-receptor stimulation. Hence, insulin altered not only fatty acid uptake, as determined by cis-parinaric and oleic acid uptake, but also altered the intracellular oleic acid esterification.

LTA cells synthesize a minor population of heparan sulfate proteoglycans (HSPGact) bearing anticoagulant heparan sulfate (HSact) with a specific monosaccharide sequence that accelerates the action of antithrombin (AT). LTA cells also synthesize a major population of heparan sulfate proteoglycans endowed with nonanticoagulant heparan sulfate (HSinact) lacking the AT-binding site. To investigate the pathway-specific features of HSPGact generation, we established a novel detergent-containing cell-free system with unlabeled and labeled microsomes from wild-type and variant LTA cells, respectively. The unlabeled microsomes provide "HSact conversion activity" that requires 3'-phosphoadenosine 5'-phosphosulfate to convert [35S]HSPGinact into [35S] HSPGact, presumably by sulfation. The reaction kinetics demonstrate that the rate of HSact synthesis is constant over the first 4 h of incubation. During this time, the rate of HSact production is linearly dependent on the amount of unlabeled LTA microsomal protein over a range of 10 to 50 microg as well as on the level of [35S]HS substrate over a range of 0.4 to 4.0 microg, microsomal protein. Compared with labeled microsomes, equivalent or slightly greater levels of HSact were generated from 35S-labeled HSPG, microsomal HS, or cell surface HS, which demonstrates that HSinact is the minimal substrate and that large amounts of HSact precursor exit the Golgi apparatus. Indeed, extensive modification of wild-type LTA cell surface [35S]HS elevated HSact content from 9 to 35%. The hypothesis that microsomal HSact conversion activity predicts the cellular rate of HSact generation was tested with wild-type or variant LTA cells in which production of HSact has been significantly altered by mutagenesis or overexpression of core protein or growth conditions. The data demonstrate that microsomal HSact conversion activity accurately reflects the cellular rate of HSact synthesis over a very wide range of conditions. The possibility that the reduced HSact generation is due to an inhibitor was excluded by mixing experiments. The possibility that reduced HSact generation is caused by decreased levels of HSact precursor was excluded as equivalent levels of HSact were formed from wild-type and variant [35S]HS. Based upon the above data, the LTA cell microsomal HSact conversion activity contains one or more limiting components that kinetically regulate the rate of cellular HSact generation and the levels of HSact precursor in HS greatly exceed HSact production.

The pathogenicity of Plasmodium falciparum is due largely to the parasite's unique ability to adhere to capillary and postcapillary venular endothelium during the second-half of the 48-hour life cycle. The resulting sequestration of infected erythrocytes (IRBC) in deep vascular beds leads to tissue hypoxia, metabolic disturbances, and organ dysfunction which characterize severe falciparum malaria. Several endothelial receptors of cytoadherence have been identified, but their clinical relevance remains controversial. In the present report, the receptor specificity of 60 clinical P falciparum isolates was determined using transfectants each expressing one of CD36, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). All isolates tested adhered to CD36 and ICAM-1, but the adherence to CD36 was at least 10-fold higher. Seven isolates adhered to E-selectin whereas none of 19 isolates adhered to VCAM-1. From a population standpoint, about 30% of IRBC in each isolate adhered to CD36, and 2% to 3% adhered to ICAM-1. The percentage adherent to E-selectin and VCAM-1 was negligible. IRBC selected on CD36 adhered almost exclusively to CD36 whereas 80% to 90% of IRBC selected on ICAM-1 could also adhere to CD36. Selected IRBC did not adhere to E-selectin or VCAM-1. These findings indicate that cytoadherence to multiple endothelial receptors is a rare occurrence with natural P falciparum isolates, but do not exclude a role for the adhesion molecules in promoting other IRBC-endothelial interactions such as rolling under flow conditions. Receptor specificity in vivo may be dictated by the ligand-receptor combination which provides the best survival potential for the parasite.

Despite the progress made on the possible functions of sterol carrier protein (SCP-2) using assays in vitro, very little is known regarding the role of SCP-2 in intact cells. To further elucidate this role, mouse L-cell fibroblasts were transfected with cDNA encoding for mouse 15 kDa or 13.2 kDa SCP-2. The data show for the first time, that SCP-2 expression increases cholesterol uptake into transfected L-cell fibroblasts. Untransfected L-cells expressed SCP-2 at levels near or below the lower limit of detectability. SCP-2 immunoreactive protein levels were 0.030 +/- 0.004% and 0.036 +/- 0.002% of total cytosolic proteins in the 15 and 13.2 kDa stable transfectants, respectively. Both the 15 and 13.2 kDa SCP-2 expressions products were found as 13.2 kDa proteins, consistent with rapid post-translational cleavage of the putative amino terminal mitochondrial targeting sequence from the 15 kDa SCP-2. The effect of expressing either form of SCP-2 on [3H]cholesterol uptake was determined. Expression of the 15 kDa form, but not the 13.2 kDa form of SCP-2, enhanced the rate and extent of [3H]cholesterol uptake compared to control or mock-transfected L-cells. The [3H]cholesterol uptake rate in 15 kDa SCP-2 expressing cells was increased 1.3-fold, while the extent of [3H]cholesterol uptake was increased 1.4-fold after 12 h of uptake compared to control L-cells. The differences in cholesterol uptake between the cells expressing the 13.2 versus the 15 kDa protein, suggest that the 15 kDa form of SCP-2 is functionally localized within the cell, while the 13.2 kDa product is not.

The role of cytosolic liver fatty acid binding protein (L-FABP) in fatty acid uptake and metabolism was examined using cultured L-cell fibroblasts transfected with the cDNA encoding for L-FABP. [3H]Oleic acid was used to determine the effects of intracellular esterification on fatty acid uptake and to determine esterified fatty acid localization to specific lipid classes. cis-Parinaric acid, a poorly esterified fatty acid, was used to determine uptake in the absence of any appreciable esterification. High-expression L-cells had a 80% and 50% greater initial uptake rate for both [3H]oleic acid and cis-parinaric acid, respectively compared to low-expression L-cells. Maximal uptake of [3H]oleic acid did not plateau because of intracellular esterification. In high-expressing cells, maximal cis-parinaric acid uptake rapidly plateaued at a level 34% higher than in low-expression cells. After 1 min of incubation, the majority of cellular [3H]oleic acid was unesterified, with the bulk of the esterified portion preferentially localized to phospholipids. After 5 and 30 min, cells expressing L-FABP esterified a significantly greater amount of [3H]oleic acid into both the neutral lipid and phospholipid fractions than did low-expression cells. L-FABP expression also selectively stimulated [3H]oleic acid incorporation into choline glycerophospholipids. Thus, L-FABP expression not only stimulated fatty acid uptake at all time points, but also stimulated intracellular esterification into specific lipid pools. These results show in detail for the first time using an intact cell culture system that L-FABP expression not only stimulated fatty acid uptake, but also increased intracellular esterification of exogenously supplied fatty acids.

A 45Ca2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45Ca2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45Ca2+ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45Ca2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45Ca2+ influx into cells expressing NR1a/NR2A receptors (IC50 = 408 microM) and NR1a/NR2B receptors (EC50 = 37.3 microM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC50 = 0.099 microM) compared with NR1a/NR2A receptors (IC50 = 164 microM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg2+ and Zn2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45Ca2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.

The mouse Hsp70.3 gene encodes the major inducible HSP70 protein and is located in close proximity to the Hsc70t gene, a testis-specific variant of the Hsp70 gene family. The two genes are arranged head-to-head, transcribed in divergent directions, and separated by approximately 600 base pairs. During transient expression analysis of the promoter for Hsc70t in mouse L cells, we have found that the Hsp70.3 coding region between -1844 and -800 relative to the transcription start site of Hsc70t possesses a silencer activity. The negative regulatory region containing this sequence also inhibited the expression from the heterologous simian virus 40 early promoter independently of orientation and position. It is possible that this silencer may not only be involved in testis-specific expression of Hsc70t but also function in down-regulation of Hsp70.3 induction.

The shellfish poison maitotoxin causes the irreversible opening of nonselective cation channels in mouse L cell fibroblasts, consistent with the action of this toxin in other cell types and the previously demonstrated existence of 28-pS voltage-insensitive nonselected cation channels that are activated by platelet-derived growth factor in these cells. Toxin-induced opening of these nonselective cation channels led to increases of intracellular calcium and secondary activation of calcium-activated potassium channel. These effects were completely dependent on influx of extracellular calcium, supporting the conclusion that the maitotoxin-activated nonselective cation channels are permeable to calcium as well as to sodium and potassium. The implication of this finding is that calcium signaling through this channel underlies its links into the growth factor response.

Mouse Ltk- cells were transfected with four different plasmids for autoinducible and highly-inducible expression of the bacterial lacZ gene and cultivated in suspension. Two selection genes, thymidine kinase (tk) and neomycin resistance (neor), were used to select the clones in both cell lines. The resulting two cell lines, designated M4 and R2, differ in that the inducible MMTV promoter from mouse mammary tumor virus (MMTV) controls glucocorticoid receptor (gr) gene and lacZ gene expression in the M4 cell line ("autoinducible"), while the constitutive rous sarcoma virus (RSV) promoter controls gr gene expression and the MMTV promoter controls lacZ gene expression in the R2 cell line ("highly-inducible"). Both cell lines were stable with respect to reproducibility of growth rate in spinner flasks and inducibility of beta-galactosidase expression. The exponential growth rate of R2 cells was slower than that of M4 cells before induction because the R2 cell line continuously expressed gr genes under the constitutive RSV promoter, and the percent reduction of exponential growth rate mainly caused by gr gene expression was about 20%. The inducibility of the M4 cell line was greater than that of the R2 cell line because in the M4 cell line MMTV promoter controlled gr and lacZ gene expression autoinducibly. Maximum induction of the M4 cell line occurred after induction with the hormone dexamethasone (Dex) at 10(-7) M, and the final beta-galactosidase content increased 400-fold after induction. The optimum conditions for inducer concentration and induction time were determined, and the highest production of beta-galactosidase occurred when Dex was added after the cell concentration had reached its maximum in batch culture. Dex (10(-9) M) is a critical inducer concentration in view of inducibility between M4 and R2 cell lines. The inducibility of R2 cell line is higher than that of the M4 cell line from 0 to 10(-9) M Dex, but the inducibility of M4 was higher than that of the R2 cell line at Dex concentrations of more than 10(-9) M.

The colon contains large numbers of endocrine cells. Insight into their physiological function is limited. This is due to the fact that no sufficient model of isolated endocrine colon cells is available. In the present study we introduce an isolated vascularly perfused colon model for in vitro studies. This model offers the advantage that it keeps the endocrine cells in their physiological orientation and environment. The gut mucosa is highly sensitive to ischemia. Therefore, a careful validation of its viability is crucial in gut organ preparations. This study demonstrates that, by utilizing an oxygenated vascular medium supplemented with 25% washed bovine erythrocytes, a perfusion of the colon is achieved for at least 1 h without obvious tissue injuries. During this time parameters such as perfusion pressure, venous lactate dehydrogenase release, glucose consumption, lactate output, oxygen consumption, perfusate loss by the preparation and morphology were analyzed. Dependent on stimulation, the endocrine L cells of the colon released glucagon-like peptide-I upon arterial perfusion of methacholine or gastrin-releasing peptide. In conclusion, a model for the isolated perfusion of the colon is introduced which is suitable for studies of endocrine colon cells.

We reported previously that murine L-929 cells expressing a human interferon (IFN)-gamma cDNA lacking a signal peptide sequence synthesize but fail to secrete human IFN-gamma and support viral replication at a reduced level. These cells also had elevated levels of IFN-inducible gene products. We show here that a similar response is seen in human cells expressing a mutated murine IFN-gamma cDNA. The ability of human IFN-gamma to induce gene expression in murine cells is shown to be due to the intracellular IFN-gamma rather than to clonal variation, induction of endogenous murine IFN, or alternative mediators of antiviral activity. We have used a murine cell line, Ltk-aprt-, which is resistant to both type I and II IFNs but responsive to combined treatment with both. Ltk-aprt- cells transfected with human IFN-gamma cDNA lacking a signal sequence support virus replication at the same level as control cells. However, unlike transfectants containing only the neoR selection gene, clones expressing the mutated human IFN-gamma gene show strong protection against viral infection and elevated levels of 2,5 A synthetase mRNA and MHC class I protein after treatment with IFN-beta alone. Reverse transcriptase-PCR rules out the induction of endogenous murine IFN expression as a mediator of these effects. Thus, expression of intracellular human IFN-gamma mimics treatment with extracellular murine IFN-gamma in permitting a synergistic response to IFN-beta. Given the inability of human IFN-gamma to bind to the murine cell-surface receptor our results show that intracellular IFN-gamma can activate certain responses independent of cell-surface binding.

Multidrug resistance (MDR) genes, which are ATP-binding cassette family genes, encode the cell surface glycoprotein, P-glycoprotein, which functions as an energy-dependent drug efflux pump. Two relevant human genes, PGY1 and PGY3, are located on human chromosome 7, and three relevant mouse genes, mdr1a, mdr1b, and mdr2, are located on mouse chromosome 5. An LMD1 cell line was established after the transfer of a 580-kb yeast artificial chromosome (YAC) clone carrying the human MDR locus into mouse L cells; the cell line was shown to have stably integrated YAC DNA in an apparent intact form. Using LMD1 cells as the parental cell line, five vincristine-resistant sublines, designated LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000, were isolated by exposure to increasing concentrations of the drug. LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000 showed 3-, 7-, 13-, 45-, and 110-fold higher resistance to the cytotoxic effects of vincristine, respectively, than their parental counterpart, LMD1. Immunofluorescence, Western blot, and Northern blot analyses revealed that the human PGY1 gene or its product was overexpressed, accompanied by gene amplification. The human PGY3 gene was also overexpressed in the LMD1-V20, LMD1-V100, and LMD1-V1000 cell lines. Southern blot and fluorescence in situ hybridization (FISH) analyses demonstrated that although essentially the entire YAC DNA was integrated in mouse genome and amplified, the endogenous mouse mdr genes were not amplified in these drug-resistant cell lines. Similar results were obtained by the analyses of vincristine-resistant cell lines isolated from four independent subclones of LMD1 cells. Thus, in contrast to their mouse counterparts, the integrated human MDR genes retained susceptibility to both gene activation and amplification, during the selection of drug-resistant mouse cell lines. The possibility that transferred YACs may retain regulatory properties observed in the cells of origin, and may have a chromatin structure that favors augmented expression, is discussed.

Differential effects of intestinal (I-FABP) or liver (L-FABP) fatty acid binding proteins on fatty acid uptake and esterification were examined using transfected mouse L-cell fibroblasts. L-FABP, but not I-FABP, expression increased the initial rate and extent of cis-parinaric acid uptake by 50 and 29%, respectively, compared to control cells. I-FABP and L-FABP expression preferentially increased [3H]-oleic acid incorporation into triacylglycerols by 5.5-fold and 3.8-fold, respectively. While both L-FABP and I-FABP increased esterification of [3H]-oleic acid into ethanolamine glycerophospholipids, these proteins had opposite effect on esterification into choline glycerophospholipids. These data show for the first time that distinct FABP differentially affect both fatty acid uptake and intracellular esterification.

In this study, we compared cholesterol efflux mediated by either high density lipoproteins (HDL3) or beta-cyclodextrins, cyclic oligosaccharides that are able to dissolve lipids in their hydrophobic core. beta-Cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, and methyl-beta-cyclodextrin at 10 mM induced the release of 50-90% of L-cell [3H]cholesterol after 8 h of incubation, with a major portion of this cholesterol being released in the first 1-2 h of incubation. The cholesterol efflux kinetics are different if cells are incubated with HDL3, which induces a relatively constant rate of release of cholesterol throughout an 8-h incubation. Cholesterol efflux to cyclodextrins was much greater than phospholipid release. To test the hypothesis that maximal efflux rate constants for a particular cell are independent of the type of acceptor, we estimated the maximal rate constants for efflux (Vmax) of cellular cholesterol from L-cells, Fu5AH cells, and GM3468A fibroblasts. The rate constant for HDL3-mediated efflux varied among cell lines in the order Fu5AH > L-cells > fibroblasts. However, these differences were not evident when cyclodextrins were used as cholesterol acceptors. The estimated Vmax values for cyclodextrin-mediated efflux were 3.5-70-fold greater than for HDL3 for the three cell lines. The very high efficiency of cyclodextrins in stimulating cell cholesterol efflux suggests that these compounds can be used in two general ways for studies of atherosclerosis: 1) as research tools to probe mechanisms of cholesterol transport and aspects of membrane structure or 2) as potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development of the atherosclerotic plaque.

Several subspecies of human high density lipoprotein (HDL) have been shown to exist, and particle size is one parameter that can be used to distinguish them. Recently, a small HDL subspecies has been described that may be a particularly efficient acceptor of peripheral cell unesterified (free) cholesterol (FC). To address the effects of particle size on the ability of HDL to remove FC from cells, homogeneous, well defined HDL particles were reconstituted (rHDL) that varied in particle diameter within the size range of human HDL particles (7-13 nm). The abilities of each of these particles to remove cellular FC from mouse L-cells and rat Fu5AH hepatoma cells were compared on the basis of their phospholipid (PL) content as well as on a per particle basis. The effect of particle size was also examined using small unilamellar vesicles (SUV) of 25 nm in diameter and large unilamellar vesicles (LUVs) of 70-180 nm in diameter. The SUV were prepared by sonication, and the LUVs were prepared by extrusion techniques. The FC efflux efficiency of these particles (in order of decreasing efficiency) was: rHDL > SUV > LUV when compared on the basis of acceptor PL content across a range of concentrations (i.e. at a given PL concentration for these three acceptor classes, smaller particles were more efficient). The FC efflux differences between the rHDL and the vesicles were not due to the absence of apolipoprotein in the vesicles. No difference was detected among the rHDL of varying size, nor was a difference detected among the LUVs of varying size when compared on the basis of PL content. When the FC efflux data for rHDL and LUVs were normalized on the basis of the number of acceptor particles present at a given PL concentration, a correlation was found between acceptor particle radius and the ability to accept cellular FC with larger particles being the most efficient. However, the dependence of the rate of FC efflux on acceptor particle size was not quantitatively the same within the rHDL and LUV classes of acceptor particles. The dependence of FC efflux on acceptor particle size may reflect differing abilities of the variously sized acceptor particles to access the region very close to the cell plasma membrane where most of the FC removal is expected to occur.

Cell surface proteoglycans, in particular those carrying heparan sulfate glycosaminoglycans, play a major role in primary attachment of herpesviruses to target cells. In pseudorabies virus (PrV), glycoprotein gC has been shown to represent the major heparan sulfate-binding virion envelope protein (T. C. Mettenleiter, L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben-Porat, J. Virol. 64:278-286, 1990). Since PrV gC is nonessential for viral infectivity in vitro and in vivo, either the interaction between virion envelope and cellular heparan sulfate is not necessary to mediate infection or other virion envelope proteins can substitute as heparan sulfate-binding components in the absence of gC. To answer these questions, we analyzed the infectivity of isogenic gC+ and gC- PrV on mouse L-cell derivatives with defects in glycosaminoglycan biosynthesis, using a rapid and sensitive fluorescence-based beta-galactosidase assay and single-cell counting in a fluorescence-activated cell sorter. Our data show that (i) in the virion, glycoprotein gC represents the only proteoglycan-binding envelope protein, and (ii) cellular proteoglycans are not essential for infectivity of PrV. Attachment studies using radiolabeled virions lacking either gC or the essential gD confirmed these results and demonstrated that PrV gD mainly contributes to binding of Pr virions to cell surface components other than proteoglycans. These data demonstrate the presence of a proteoglycan-independent mode of attachment for Pr virions leading to infectious entry into target cells.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.

Using a clonal cell line that stably expresses the murine luteinizing hormone receptor (LHR 11/6 cells), we studied the molecular mechanisms of agonist-induced desensitization of the luteinizing hormone/chorionic gonadotropin-responsive adenylyl cyclase. Exposure of transfected cells to human chorionic gonadotropin (hCG) resulted in a dose-dependent loss of maximal hCG-stimulable adenylyl cyclase activity without a significant shift to the right of the dose-response curve to hCG. This rapid uncoupling of the LH receptor from the cellular adenylyl cyclase system was not accompanied by internalization of receptor sites. A 6-h exposure to hCG led only to minor (ca. 25%) loss of membrane binding sites. The dose-response curve to hCG was not altered by pretreating cells with 8-Br-cAMP or prostaglandin E1. These findings, and the observation that hCG-induced desensitization can still be monitored at Mg2+ concentrations in the assay as high as 10 mM, preclude a significant contribution of protein kinase A to LH receptor uncoupling. The murine LH receptor not only stimulates adenylyl cyclase but also phospholipase C and probably protein kinase C (PKC) via diacylglycerol. Activation of PKC by 4 beta-phorbol 12-myristate 13-acetate failed to desensitize. When PKC was down-regulated hCG could still exert a maximal desensitizing effect. It is concluded that in LHR 11/6 cells there is no evidence for a major role of PKC in homologous desensitization. Thus, it is likely that a second messenger-independent kinase, such as beta-adrenergic receptor kinase, or a different, as yet unknown mechanism is involved in the agonist-induced desensitization of the LH receptor.

The binding of insulin-like growth factor II (IGF II) to the mannose 6-phosphate (M6P)/IGF II receptor has previously been reported to induce the activation of trimeric G(i)2 proteins by functional coupling to a 14-amino acid region within the cytoplasmic receptor domain (Nishimoto, I., Murayama, Y., Katada, T., Ui, M., and Ogata, E. (1989) J. Biol. Chem. 264, 14029-14038). In the present study, we examined further the potential functional coupling of G-proteins with the human M6P/IGF II receptor and mutant receptors lacking the proposed G-protein activator sequence. IGF II treatment of mouse L-cells expressing either wild type or mutant M6P/IGF II receptors failed to attenuate the pertussis toxin-catalyzed modification of a 40-kDa protein or enhance GTPase activity. In broken L-cell membranes expressing wild type or mutant M6P/IGF II receptors, 30 nM IGF II also failed to affect the pertussis toxin substrate activity. By using phospholipid vesicles reconstituted with human wild type or mutant M6P/IGF II receptors and pertussis toxin-sensitive G-proteins, no stimulation of GTP gamma S binding to or GTPase activity of G(i)2, G(o)1, or G(i)/G(o) mixtures were observed in response to 1 microM IGF II. Furthermore, in vesicles containing purified wild type M6P/IGF II receptors and monomeric G alpha o1 or G alpha i2 and beta gamma dimers no effects of IGF II on GTP gamma S binding could be detected. However, when vesicles reconstituted with M6P/IGF II receptors and G(i)2 proteins were incubated with 100 microM mastoparan GTP gamma S binding was stimulated and GTPase activity was increased significantly. These results indicate that the human M6P/IGF II receptor neither interacts with G-proteins in mouse L-cell membranes nor is coupled to G(i)2 proteins in phospholipid vesicles. This study suggests strongly that the M6P/IGF II receptor does not function in transmembrane signaling in response to IGF II.