What Is an L-Cell and How Do We Study the Secretory Mechanisms of the L-Cell?

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.

Middle Ear "Adenoma": a Neuroendocrine Tumor with Predominant L Cell Differentiation.

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.

Profiling of extracellular matrix and cadherin family gene expression in mouse feeder layer cells: type VI collagen is a candidate molecule inducing the colony formation of epithelial cells.

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.

Regulation of MAP kinase-directed mitogenic and protein kinase B-mediated signaling by cannabinoid receptor type 1 in skeletal muscle cells.

OBJECTIVE: The endogenous cannabinoid (or endocannabinoid) system (ECS) is part of a central neuromodulatory system thought to play a key role in the regulation of feeding behavior and energy balance. However, increasing evidence suggests that modulation of the ECS may also act to regulate peripheral mechanisms involved in these processes, including lipogenesis in adipose tissue and liver, insulin release from pancreatic beta-cells, and glucose uptake into skeletal muscle. It was recently shown that cannabinoid receptor type 1 (CB1) and type 2 (CB2), both key components of the ECS, are expressed in human and rodent skeletal muscle. However, their role in modulating insulin sensitivity in this metabolically active tissue has yet to be determined. Our aim was to establish the role, if any, of these receptors in modulating insulin sensitivity in skeletal muscle cells. RESEARCH DESIGN AND METHODS: Cultured skeletal muscle cells were exposed to CB1 and/or CB2 pharmacological agonists/antagonists/inverse agonists, and the resulting effects on insulin-regulated phosphatidylinositol 3 kinase (PI 3-kinase)-protein kinase B (PKB) and extracellular signal-related kinases 1/2 (ERK1/2)-directed signaling were determined. RESULTS: Here, we report that modulating the activity of the ECS in skeletal muscle regulates both insulin-dependent mitogen-activated protein (MAP) kinase (ERK1/2) and the canonical PI 3-kinase/PKB signaling pathways. We show that pharmacological activation or inhibition of CB1 receptor activity exerts a differential effect with regard to MAP kinase- and PKB-directed signaling. CONCLUSIONS: Our study provides evidence that signaling via cannabinoid receptors can significantly modulate mitogenic and metabolic signaling in skeletal muscle with important implications for muscle growth and differentiation as well as the regulation of glucose and lipid metabolism.

What is known, new and controversial about GLP-1? Minutes of the 1st European GLP-1 Club Meeting, Marseille, 28-29 May 2008.

The first antidiabetic agent was a hormone--insulin--and ever since, all therapeutic strategies have been based on the synthesis of chemical compounds to bind its receptors or transcription factors, or to trigger its intracellular mechanisms. Eighty years on, new therapeutic molecules are available for the treatment of diabetes and, again, are based on a hormone--glucagon-like peptide-1 (GLP-1). Whereas the theoretical benefit of insulin is based on normalization of functional physiology, therapeutic strategies based on GLP-1 aim to increase the circulating concentration of a natural component--the hormone GLP-1. There are two strategies for increasing GLP-1 plasma concentrations: replace the hormone with a long-acting analogue or molecule with a longer half-life; and prevent its degradation by inhibiting its natural protease, dipeptidyl peptidase IV (DPPIV). Although numerous clinical trials have been carried out and vast amounts of data are available, the mechanisms through which GLP-1-based therapy reduces blood glucose in diabetic patients remain unclear. Thus, it is essential to ask the right questions and to design appropriate clinical trials and experiments to increase our understanding of the mode of action of GLP-1-based therapy. For this reason, in the spring of 2008, expert scientists and clinicians in the field of GLP-1 got together for an intensive debate on the subject at the first meeting of the European Club for the study of GLP-1, held in Marseille. The subject of the round table discussions was: what is known, new and controversial about GLP-1? During these discussions, numerous facts and controversies were reevaluated, and revealed that several long-held, dogmatic beliefs have never been fully and scientifically established. These points are detailed here in these minutes of the landmark meeting.

Blockade of tumor necrosis factor (TNF) receptor type 1-mediated TNF-alpha signaling protected Wistar rats from diet-induced obesity and insulin resistance.

TNF-alpha plays an important role in the pathogenesis of obesity and insulin resistance in which the effect of TNF-alpha signaling via TNF receptor type 1 (TNFR1) largely remains controversial. To delineate the role of TNFR1-mediated TNF-alpha signaling in the pathogenesis of this disorder, a TNFR1 blocking peptide-Fc fusion protein (TNFR1BP-Fc) was used for the present study. Wistar rats were fed a high-fat/high-sucrose (HFS) diet for 16 wk until obesity and insulin resistance developed. In comparison with increased body weight and fat weight, enlarged adipocytes, and hypertriglyceridemia in the obese state, the subsequent 4-wk treatment with TNFR1BP-Fc resulted in significant weight loss characterized by decreased fat pad weight and adipocyte size and reduced plasma triglycerides. Furthermore, obesity-induced insulin resistance, including hyperinsulinemia, elevated C-peptide, higher degree of hyperglycemia after glucose challenge, and less hypoglycemic response to insulin, was markedly improved, and the compensatory hyperplasia and hypertrophy of pancreatic islets were reduced. Interestingly, treatment with TNFR1BP-Fc markedly suppressed systemic TNF-alpha release and its local expression in pancreatic islets and muscle and adipose tissues. In addition, blockage of TNFR1-mediated TNF-alpha signaling in obese rats significantly enhanced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in the muscle and fat tissues. Our results strongly suggest a pivotal role for TNFR1-mediated TNF-alpha signaling in the pathogenesis of obesity and insulin resistance. Thus, TNFR1BP-Fc may be a good candidate for the treatment of this disease.

Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes.

Polyethylenimine (PEI) is one of the most efficient nonviral vectors for gene therapy. The aim of this study was to investigate the role of endocytosis in the transfection of synchronized L929 fibroblasts by PEI/DNA complexes. This was performed by confocal microscopy and flow cytometry, using the endocytosis marker FM4-64 and PEI/DNA complexes labeled either with the DNA intercalator YOYO-1, or with fluorescein covalently linked to PEI. Endocytosis appeared as the major if not the sole mode of entry of the PEI/DNA complexes into the L929 cells. The complexes followed a typical fluid phase endocytosis pathway and were efficiently taken up in less than 10 min in endosomes that did not exceed 200 nm in diameter. Later, the localization of the complexes became perinuclear and fusion between late endosomes was shown to occur. Comparison with the intracellular trafficking of the same complexes in EA.hy 926 cells (W.T. Godbey, K. Wu, A.G. Mikos, Proc. Natl. Acad. Sci. USA 96 (1999)) revealed that endocytosis of PEI/DNA complexes is strongly cell-dependent. In L929 cells, escape of the complexes from the endosomes is a major barrier for transfection. This limited the number of transfected cells to a few percent, even though an internalization of PEI/DNA complexes was observed in most cells. In addition, the entry of the complexes into the nucleus apparently required a mitosis and did not involve the lipids of the endosome membrane. This entry seems to be a short-lived event that involves only a few complexes.

Recognition of chlamydial antigen by HLA-B27-restricted cytotoxic T cells in HLA-B*2705 transgenic CBA (H-2k) mice.

OBJECTIVE: The association of reactive arthritis (ReA) with HLA-B27 and the presence of bacterial antigen in joints with ReA suggest that bacterial peptides might be presented by the HLA-B27 molecule and thus stimulate CD8 T cells. This study was performed to investigate the B27-restricted cytotoxic T lymphocyte (CTL) response to Chlamydia trachomatis, using the model of HLA-B27 transgenic mice. METHODS: CBA (H-2k) mice homozygous for HLA-B*2705 and human beta2-microglobulin expression were immunized with C trachomatis or with the chlamydial 57-kd heat-shock protein (hsp57) coupled to latex beads. Cytotoxicity of lymphocytes from in vivo-primed transgenic mice was tested against C trachomatis-infected targets. Blocking experiments were performed with monoclonal antibodies (MAb) against class I major histocompatibility complex molecules. RESULTS: A Chlamydia-specific lysis of both B27-transfected and nontransfected target cells was observed. This response could be inhibited by anti-B27 and anti-H2 MAb. CTL from mice immunized with hsp57 were not able to lyse Chlamydia-infected target cells, and Chlamydia-specific CTL could not destroy targets loaded with hsp57. CONCLUSION: These results suggest the existence of at least 2 CTL populations in this mouse model: one recognizing peptide of bacteria-infected cells restricted by HLA-B*2705 and the other recognizing peptide of bacteria-infected cells restricted by the murine H-2Kk molecule. It does not appear that hsp57 is a major target for the CD8 T cell response directed against Chlamydia. This animal model opens the way for identifying bacterial epitopes presented by HLA-B27, and might thus help to clarify the pathogenesis of B27-associated diseases.

Interactions of tunicate immunomodulatory proteins with mammalian cells.

The ability of a tunicate immunomodulatory protein, tunIL1, to interact with mammalian cells was investigated. TunIL1 is known to regulate inflammatory defence reactions in tunicates and to stimulate the proliferation of mammalian thymocytes. In the current study, tunIL1 was shown to enchance L929 fibroblast proliferation, induce IL-2 secretion from human mononuclear cells and enhance IL-2 receptor expression by EL-4 murine lymphoma cells. These biological activities are comparable with those of the mammalian inflammatory cytokine, IL-1. However, tunIL1 does not appear to stimulate its effects on mammalian cells by interacting with cell surface receptors in a manner analogous to mammalian IL-1. TunIL1 cannot block the binding of anti-IL-1 receptor antibodies to EL-4 cells, nor can anti-IL-1 receptor antibodies inhibit the capacity of tunIL1 to stimulate thymocyte proliferation. This indicates that tunIL1 does not induce its IL-1-like activities via structural homology to mammalian IL-1.

Direct and indirect mechanisms of repression participate in suppression of T-cell-specific gene expression in T x L-cell hybrids.

Expression of tissue-specific genes can be altered upon fusion of mammalian cells of different types. To resolve the genetic basis of this phenomenon and to identify components of the regulatory circuits that are involved, we have established a series of somatic cell hybrids between mouse T cells and L cells. These hybrids have an unusual and interesting phenotype. Unlike many hybrid cells studied, in which the expression of an entire set of tissue-specific genes was coordinately extinguished, in our T x L-cell hybrids only two out of seven T-cell-restricted genes were completely extinguished, whereas the other genes were repressed to various degrees. These hybrids extinguish the production of TCR beta and Thy-1 mRNA, repress the expression of TCR alpha, GATA-3, TCF-1, and LEF-1 genes to different extents, exhibit small changes in the level of CD3-epsilon mRNA, and continue to express the fibroblast-specific fibronectin gene, and the ets-1 gene. In this study we have evaluated for the first time the molecular mechanisms that underlie the repression of TCR alpha and TCR beta chain genes in T x L-cell hybrids. We have shown that multiple repression mechanisms, both direct and indirect, contribute to TCR alpha and TCR beta suppression. Repression of the expression of these genes correlated not only with the downregulation of GATA-3, TCF-1, and LEF-1 transcription factor expression, and with a change in the chromatin structure, but more importantly, with the activation of the silencer activity. Our study provides evidence for the existence of at least two negatively regulating elements, located at the TCR alpha enhancer-containing fragment and at the silencer region, which are active in our hybrid cells. We have shown that there was no correlation between the levels of GATA-3, TCF-1, and LEF-1 expression versus the level of TCR alpha mRNA in the independent hybrids. In contrast, both the silencer activity and the ability of the TCR alpha enhancer to downregulate thymidine kinase (TK) promoter activity were found to be in an inverse correlation with the ability of the different hybrid cells to express TCR alpha mRNA.

Voltage sensing by fluorescence resonance energy transfer in single cells.

A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nernst equation. A voltage-sensitive fluorescent readout is created by labeling the extracellular surface of the cell with a second fluorophore, here a fluorescently labeled lectin, that can undergo energy transfer with the membrane-bound sensor. Fluorescence resonance energy transfer between the two fluorophores is disrupted when the membrane potential is depolarized, because the anion is pulled to the intracellular surface of the plasma membrane far from the lectin. Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl is n-hexyl and n-decyl (DiSBA-C6-(3) and DiSBA-C10-(3), respectively) can function as donors to Texas Red labeled wheat germ agglutinin (TR-WGA) and acceptors from fluorescein-labeled lectin (FI-WGA). In voltage-clamped fibroblasts, the translocation of these oxonols is measured as a displacement current with a time constant of approximately 2 ms for 100 mV depolarization at 20 degrees C, which equals the speed of the fluorescence changes. Fluorescence ratio changes of between 4% and 34% were observed for a 100-mV depolarization in fibroblasts, astrocytoma cells, beating cardiac myocytes, and B104 neuroblastoma cells. The large fluorescence changes allow high-speed confocal imaging.

Cell surface P- and E-selectin support shear-dependent rolling of bovine gamma/delta T cells.

The vascular selectins P- and E-selectin are inducible adhesion proteins expressed by endothelial cells that have been shown to support shear-dependent rolling of myeloid cells. This interaction is thought to be a prerequisite event for subsequent steps, such as tight adhesion/aggregation and transendothelial cell migration, involved in the accumulation of leukocytes into tissues. Certain lymphocyte subsets have also been shown to bind the vascular selectins, but the importance of this interaction in mediating shear-dependent rolling, as described for myeloid cells, has not been demonstrated. We expand on our earlier observation that bovine gamma/delta T cells bind E-selectin by showing that this interaction leads to a reproducible rolling event in assays done under shear forces that approximate those that occur in vivo. E-selectin, expressed by L cell transfectants or cytokine-stimulated human and bovine endothelial cells, equally supports the shear-dependent rolling interaction. The lymphocyte adhesion proteins L-selectin, CD44, and CD2 do not contribute to this event. Neuraminidase treatment of the gamma/delta T cells or addition of EDTA to the assay completely blocks the rolling interaction. We further show for the first time that P-selectin expressed by thrombin-activated platelets or a soluble P-selectin/human Ig chimera specifically binds gamma/delta T cells. The P-selectin interaction is similar to the rolling event mediated by E-selectin--it requires divalent cations and sialic acid on the lymphocyte, it lacks involvement of L-selectin and CD44, and rolling occurs under physiologic shear conditions. These results provide the documentation that the vascular selectins can support shear-dependent rolling of a lymphocyte subset and that P-selectin mediates the adhesion of gamma/delta T cells.

In vitro bioassay for human serum follicle-stimulating hormone (FSH) based on L cells transfected with recombinant rat FSH receptor: validation of a model system.

FSH plays a central role in normal reproductive function, i.e. control of follicular maturation in the female and initiation and maintenance of spermatogenesis in the male. The effects of FSH are mediated by its interaction with a specific receptor that belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors. Due to the microheterogeneity of gonadotropins, measurement of immunoreactivity does not necessarily reflect their bioactivity. Mutations in gonadotropin beta-subunits, which affect bioreactivity and/or immunoreactivity of gonadotropins, have been described as causes of infertility, thus highlighting the need for rapid and convenient methods to measure bioactivity. To establish a model system for recombinant in vitro bioassays for FSH that would obviate the use of live animals, we developed a strategy for efficient expression of the rat FSH receptor (FSHR) in L cells. A cell line, FSHR 7/12, was developed that bound [125I]FSH with high affinity (Kd 1.42 nM) and responded to human FSH with an increase in cAMP accumulation. Untreated human serum was found to have an unspecific inhibitory effect on cAMP formation. This effect could be thoroughly avoided by mild heating (10 min at 56 C) of serum samples before addition to cells without detectable loss of FSH immunoactivity or bioactivity. Studies on the hormone-sensitive adenylyl cyclase system of transformed FSHR 7/12 cells and of the parental Ltk- cells showed that the cellular response to FSH was highly specific. Using a parallel line assay design, FSHR 7/12 cells were used to validate a novel recombinant in vitro bioassay relying on intracellular cAMP accumulation as a readout system. Up to 10% of serum could be added to the incubation buffer without leading to nonparallelism to the standard curve. When 70 serum samples of male patients attending an infertility clinic were analyzed, the novel assay system displayed high sensitivity and a close correlation (r > 0.8; P < 0.01) to the established rat Sertoli cell aromatase bioassay and to a highly specific fluoroimmunoassay. When sera of 25 normal menstruating women were analyzed for FSH bioactivity at different stages of the menstrual cycle, a midcycle FSH peak followed by a decline in the late luteal phase could be discerned. The analysis of 26 serum samples of postmenopausal women revealed a close correlation between FSH values obtained by the novel in vitro bioassay and by a fluoroimmunoassay (r = 0.90; P < 0.01). Thus, the present in vitro bioassay represents a sensitive, rapid, and convenient model system to measure bioactive FSH in human serum.

Proliferation of human leukemic pre-B cells induced by human bone marrow stromal cells and murine fibroblasts.

Aim of the study was the establishment of a standardized in vitro culture system for studies on proliferation and differentiation of human leukemic pre-B cells. Coincubation with human stromal cells led to a significantly higher proliferation of the cytokine-sensitive leukemic pre-B cell line BLIN-1. Clones from the murine fibroblast cell line L929 provided identical results. Coincubation with the murine cells also resulted in a significantly higher numbers of viable cells in 5 of 8 patient samples with newly diagnosed B lineage ALL. The results show that in vitro bone marrow stromal cells can be substituted by murine fibroblasts and form the basis for a simpler and more reproducible assay system.

Extracellular domain of pemphigus vulgaris antigen (desmoglein 3) mediates weak homophilic adhesion.

Pemphigus vulgaris antigen is in the cadherin supergene family. We hypothesized that the extracellular domain of pemphigus vulgaris antigen might mediate homophilic cell adhesion because 1) the originally described cadherins (e.g., E-cadherin) mediate this type of adhesion, 2) pemphigus vulgaris antigen is localized in desmosomes that are cell adhesion junctions, and 3) autoantibodies in pemphigus vulgaris patients cause loss of cell adhesion. To test this hypothesis we used a system developed for E-cadherin that, when transfected into L cells (mouse fibroblasts), has been shown to cause aggregation. Because this aggregation requires the cytoplasmic domain of E-cadherin to bind to catenins, we made a chimeric cDNA construct that encodes the extracellular domain of pemphigus vulgaris antigen and the cytoplasmic domain of E-cadherin. Analysis by immunofluorescence and flow cytometry with pemphigus vulgaris sera indicated that the pemphigus vulgaris antigen extracellular domain of this chimeric molecule (PVEC) was expressed on the cell surface of transiently transfected cells and permanently transfected L-cell clones. Immunoprecipitation of the chimeric molecule from extracts of these clones showed that the E-cadherin cytoplasmic domain bound catenins. Surprisingly, these L-cell clones displayed only slight aggregation compared to an L-cell clone transfected with E-cadherin. This weak aggregation was, however, specific and homophilic, as determined by cell sorting of only PVEC transfectants into aggregates from mixtures of PVEC and neomycin resistance gene transfectants, one of which was labeled with a fluorescent dye. We conclude that the extracellular domain of pemphigus vulgaris antigen mediates weak homophilic adhesion and is not interchangeable in function with the extracellular domain of E-cadherin.

Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin.

Langerhans cells (LC) are the principal accessory cells present in epidermis. Because LC have limited capacity for self-renewal, epidermis is continually repopulated by as-yet uncharacterized bone marrow-derived LC progenitors. In addition, although LC persist in epidermis for extended periods, LC are induced to migrate from skin to regional lymph nodes after antigen exposure. To begin to elucidate mechanisms involved in LC trafficking, we characterized LC-keratinocyte (KC) interactions. Here we report that fresh murine LC express cadherins, and that LC adhere to KC in vitro through E-cadherin. Cultured LC (which may bear a phenotypic and functional relationship to LC that have migrated to lymph nodes) express lower levels of E-cadherin and exhibit decreased affinity for KC. These results suggest that expression of E-cadherin by LC promotes persistence of these cells in epidermis, and that cadherins may play important and unanticipated roles in interactions between leukocytes and epithelia.

[The participation of endonuclease in the formation of extrachromosomal DNA and the possible mechanisms of the occurrence of gene amplification]. / Uchastie éndonukleazy v orbazovanii ékstrakhromosomnoi DNK i vozmozhnye mekhanizmy vozniknoveniia amplifikatsii genov.

We examined the extrachromosomal DNA (exDNA, Hirt fraction) in ethidium bromide sensitive and resistant cells of line L929. The exDNA amount is greater in the latter. The amount of exDNA in L929 cells makes 0.19% of the total cellular DNA; the exDNA amounts in cells, resistant to 5 and 50 micrograms/ml ethidium bromide are 0.22 and 0.33%, resp. Using labelling by BudR, it is shown that approximately 16% exDNA in L cells constituted amplified sequences to be excreting to the culture medium. The Zn-independent endogenous nuclease is activated in the resistant cells. The treatment with cycloheximide (50 micrograms/ml) resulted in the increase in the exDNA amount and in the activation of Zn-independent endonuclease. The data obtained suggested that the activation of Zn-independent endonuclease may lead to the increase in the exDNA amount and determine presumably a high rate of cell adaptability to environmental conditions.

Absence of HSP28 synthesis and phosphorylation during the development of chronic thermotolerance in murine L929 cells.

We investigated the correlation between chronic thermotolerance development and phosphorylation, synthesis, or expression of the HSP28 family in murine L929 cells. Chronic thermotolerance developed during heating at 41.5 degrees C as indicated by a biphasic survival curve. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, we failed to detect HSP28 synthesis during chronic heating by using two-dimensional polyacrylamide gel electrophoresis. The lack of HSP28 synthesis was also confirmed in acute thermotolerance. Similar results were observed in NIH 3T3 cells. Although Southern blots clearly demonstrated the presence of the HSP28 gene in genomic DNA, Northern blots failed to demonstrate its expression. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, were stimulated during chronic heating at 41.5 degrees C in L929 cells. These results suggest that HSP28 synthesis and its phosphorylation are not required to develop both chronic and acute thermotolerance in L929 cells.

The stimulatory effect of actinomycin D on avian reovirus replication in L cells suggests that translational competition dictates the fate of the infection.

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.

Avian reovirus S1133 can replicate in mouse L cells: effect of pH and cell attachment status on viral infection.

Previous reports have suggested that avian reovirus S1133 fails to replicate in mouse L cells. In this article, we report that replication does occur under certain culture conditions. The avian reovirus was found to grow in mouse L cells at pH 6.4 and 7.2 but not at pH 8.2. Culture medium with a basic pH directly inhibited viral transcription and genome replication. As a result, viral protein synthesis was also affected. At permissive pH levels, avian reovirus grew better in monolayers than in suspension cultures of L cells because of the influence of cell attachment status on viral macromolecular synthesis. Our results not only show that avian reovirus can replicate in mouse L cells but also help to explain why it did not in previous studies.