The TRH neuronal phenotype forms embryonic cell clusters that go on to establish a regionalized cell fate in forebrain

How neurons diversify in developing brain to produce discrete cell fates in their appropriate regions remains a fundamental question. Embryonic Xenopus was previously used to identify juxtaposed embryonic cells that first express proopiomelanocortin mRNA in forebrain and pituitary, supporting the idea that this neuropeptide phenotype is induced locally. (Hayes and Loh, 1990, Development 110:747–757). To begin to examine how a more widespread population of forebrain cells is set up, the present focus is on the thyrotropin-releasing hormone (TRH) phenotype. Serial section in situ hybridization histochemistry produced the unexpected finding that the adult-like TRH system spanning forebrain and comprising over six different telencephalic and diencephalic nuclei, is preceded by an embryonic TRH cell population that is initially localized and then highly regionalized in the area from which the adult pattern develops. Thus, the first TRH cells, detected in vivo after 35 h (stage 29/30), were confined to discrete anterior or posterior bilateral clusters in embryonic forebrain or hindbrain. Thereafter, the TRH cell clusters in diencephelon, but not hindbrain, expanded to form rows, extending anteriorly into telencephalon and bifurcating posteriorly around the infundibulum. By 80 h (stage 42), after extensive brain morphogenesis, these forebrain rows showed regional differences in levels of TRH and mRNA corresponding to the specific brain nuclei that have been shown to contain TRH cells in adult. These findings show that subsets of phenotype-specific forebrain cell first form a regionalized neuronal cell fate before distinct brain nuclei form. This is turn points to the testable hypothesis in Xenopus that certain neuronal cell fates in forebrain may be dictated by cell lineage or local induction. 1994 John Wiley & Sons, Inc.     

Effects of TRH on cell proliferation of rat pituitary cells (GH3)

Summary Chronic treatment (more than 3 d) of GH₃ cells, cloned rat pituitary cells producing prolactin, with 100 nM TRH resulted in a 41% reduction in the rate of cell growth in a medium containing 0.5% fetal bovine serum. These effects of TRH appeared both in the medium containing a higher concentration of serum and in that containing six growth factors, i.e. insulin, transferrin, parathyroid hormone, fibroblast growth factor, triiodothyronine, and multiplication-stimulating activity (MSA) instead of serum. TRH stimulated prolactin production by GH₃ cells in a dose-dependent manner both in the serum-supplemented and serum-free media. On the other hand, TRH, at 1 nM, elicited a 130% stimulation in the cellular growth, whereas, at concentrations of more than 10 nM, it inhibited the growth significantly. In the defined culture system, it was demonstrated that TRH stimulated prolactin production in the presence or absence of six growth factors, whereas its inhibitory effects on cellular growth appeared only in the presence of MSA regardless of the presence or absence of the other five factors. Furthermore, it was shown that a dose-dependent stimulatory effect of MSA on the growth of GH₃ cells was suppressed by TRH. TRH exhibited only a stimulatory effect on cellular growth in the medium containing the five factors other than MSA. In conclusion, TRH could inhibit cell growth of GH₃ in the presence of MSA in the defined medium or MSA-like factor(s) in the serum-supplemented medium.     

Disruption of the Plasma Membrane Integrity by Cholesterol Depletion Impairs Effectiveness of TRH Receptor-Mediated Signal Transduction via Gq/G11α Proteins

We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G_q/11α proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G₁₁α. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G_q/11α was assessed by agonist-stimulated [³⁵S]GTPγS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca²⁺ response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G_q/11α proteins.     

Analysis of the flt-1 Gene Encoding a ECM Protein, Flectin, in Caenorhabditis elegans

Flectin is a new type of extracellular matrix protein and its function was suggested to provide a micro-environment of great elasticity. The C. elegans genome database revealed the presence of a flectin homologue, flt-1, which shows approximately 40% similarity (20% identity) to chick flectin. Here we propose a new gene structure for the flt-1 based on our experiments and the partial cDNA clones obtained from Y. Kohara and further suggest that the previous gene prediction is incorrect. FLT-1 is shown to be expressed in various neurons, hypodermal cells, distal tip cells and vulva epithelial cells. Immunostaining results with anti-FLT-1 antibody, further confirm the FLT-1 expression in vulva epithelial cells. The lipophilic dye, DiI, was used to identify the head neurons expressing GFP and results indicated that none of the head neurons expressing GFP are the 6 chemosensory neurons. In order to determine the function of flt-1 gene, RNA-mediated interference (RNAi) experiments were conducted.     

Localization of thyrotropin-releasing hormone-and insulin-immunoreactivity in the pancreas of neonatal rats after injection of streptozotocin at birth

Summary Streptozotocin treatment at birth induces, in the pancreas of rats, first depletion of insulin and thyrotropin-releasing hormone and then early regeneration of cells and insulin, but not TRH. This study was undertaken to investigate whether the reduction in pancreatic TRH content can be associated with changes in the intensity and the distribution of TRH-immunoreactivity, and to follow the pattern of regeneration of cells through insulin- and TRH-immunoreactivity.In control animals, strong TRH-immunoreactivity was seen in insulin-containing cells on days 1–4 after birth. At day 7, the TRH-immunoreactivity was already decreased. In contrast, insulin-immunoreactivity was present throughout the neonatal period. A sparse population of cells near ducts also contained both TRH- and insulin-immunoreactivity at 1–2 days age.In streptozotocin-treated animals, TRH-immunoreactivity is found only in a few scattered insulin-containing cells in altered islets on days 1–4. Near the ducts, there were new insulin-containing cells which did not contain TRH. From day 7 regeneration of endocrine cells was characterized by new, typical islets, but these contained insulin-, but not TRH-immunoreactivity. These findings suggest a differential control of the biosynthesis of insulin and TRH within the pancreas.     

Establishment and cryopreservation of a cell line derived from caudal fin of endangered catfish Clarias dussumieri Valenciennes, 1840

We describe a new cell line, Clarias dussumieri fin (ClDuF), from the caudal fin of C. dussumieri using the explant technique followed by cryopreservation. The cryopreserved CiDuF cells were validated for quality and other characteristics. They showed typical epithelial morphology in vitro and epithelial cells outgrew their fibroblast cells after the fifth passage. ClDuF cells had a characteristic sigmoid curve with population doubling in 24 h. Immunotyping of the ClDuF cells against cytokeratin suggested the epithelial lineage. Chromosome analysis showed normal diploid (2n = 50) numbers and the cells did not contain any contamination, including Mycoplasma and other microbes. Partial sequencing of fragments of mitochondrial 16s rRNA and COI genes of ClDuF confirmed that the cell line was initiated from C. dussumieri. Cells at the 10th and 25th passages had more than 80% and 70% viability in the culture, respectively, after 6 months of storage at LN₂. These ClDuF cells were morphologically identical to the cells before freezing and the genetic resource of C. dussumieri was preserved. The species-specific cells can serve as a valuable source for virus isolation, conservation and cloning of somatic cells.     

Some lectin binding properties of the tongue of the mole rat,Nannospalax xanthodon

We investigated carbohydrate residues on the epithelial surface, in the epithelial cells and in gland cells of the tongue of the mole rat using histochemical methods. We used horseradish peroxidase-conjugated lectins from Helix pomatia (HPA), Arachishypogaea (PNA), Ulexeuropaeus (UEA I), Canavaliaensiformis (Con A). The most intense reactivity was observed in the keratin layer with HPA, UEA I and Con A, and in the epithelial cells with UEA I and Con A. In the glands, we found strong reactivity in serous cells with HPA and Con A, and in mucous cells with HPA and UEA I. PNA did not bind to epithelial or gland cells. Consequently, GlcNAc, fucose and α-D-mannose terminal glycoconjugates are distributed widely; GalNAc terminal glycoconjugates appeared in small amounts.     

The effects of the adrenoreceptor agonists phenylephrine and isoproterenol on the intracellular ion concentrations of branchial epithelial cells of brown trout (Salmo trutta L.)

Brown trout were fitted with indwelling, intraperitoneal catheters and injected with 4–6 mol · kg^-1 of the -receptor agonist phenylephrine or the -receptor agonist isoproterenol. The intracellular concentrations of sodium, chlorine, potassium and phosphorus in the pavement epithelial cells and the mitochondria-rich cells of the branchial epithelium were measured by X-ray microanalysis 1 h after the injection of the adrenoreceptor agonists. Injection with phenylephrine resulted in a significant increase in intracellular chlorine and potassium in mitochondria-rich cells and a significant but relatively smaller increase in chlorine in pavement epithelial cells. Injection with isoproterenol resulted in a significant increase in sodium and chlorine concentration in pavement epithelial cells and a significant decrease in potassium concentration. The only significant effect of isoproterenol injection on mitochondria-rich cells was a decrease in intracellular chlorine concentration. The results suggest that these adrenoreceptor agonists have a direct effect on the influx of Na⁺ and Cl^- across the branchial epithelium. These effects may be a mechanism for acid-base regulation during the severe stress conditions that elicit catecholamine release in vivo. These results corroborate previous studies using X-ray microanalysis which suggested that pavement epithelial cells are the sites of Na⁺ uptake in freshwater fish whilst Cl^- uptake occurs via mitochondria-rich cells.Abbreviations LTSEM low-temperature scanning electron microscope - MR cells mitochondria-rich cells - PE cells pavement epithelial cells - XRMA X-ray microanalysis     

Rapid obtention of stable,bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

Background  

Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors.     

PERP,a host tetraspanning membrane protein,is required for Salmonella‐induced inflammation

Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast‐two‐hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP‐22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface.     

Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE^−/−) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1β at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes—and not epithelial cells—and was dependent on NLRP3 inflammasome activation and IL1β-signalling. In females, CFA-induced TSLP was independent of IL1β and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR^−/− mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE^−/− and ApoE^−/−/TSLPR^−/− mice with either CFA/IFA or PBS. ApoE^−/− mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE^−/−/TSLPR^−/− mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.     

MICA, a new polymorphic HLA-related antigen, is expressed mainly by keratinocytes, endothelial cells, and monocytes

 MICA is a new polymorphic gene in the HLA region expressed in epithelial cell lines and gastrointestinal epithelium. Little is yet known about the MICA protein, and the pattern of its expression by freshly isolated cells has not been established. In the present experiments, we used antibodies raised in rabbits against α1 and α2 domain-peptides to study the expression of MICA. By western blot and immunoprecipitation, we detected a band of 62 000 M _r in various cell lines (THP-1, U937, HeLa, A431, Raji, MOLT-4, and HUV-EC-C) and in freshly isolated keratinocytes, endothelial cells, and monocytes but not in CD4⁺ and CD8⁺ T cells, and CD19⁺ cells (B lymphocytes). It was not possible to up-regulate the expression of MICA in different cells by stimulation with γ-interferon, but the expression of MICA was induced in phytohemagglutinin-stimulated T cells. We confirmed that MICA is expressed at the cell surface by flow cytometry. Results of immunoprecipitation studies of β₂-microglobulin (β₂m)- or MICA-depleted, metabolically labeled HeLa cells indicated that MICA was not associated with β₂m. Although the function of MICA is still unknown, its restricted pattern of tissue expression, the fact that it is expressed on the cell surface, and its polymorphic nature suggest that this new molecule, encoded close to HLA class I, may play a role in the interaction between epithelial cells and cells of the immune system. Received: 21 May 1997 / Revised: 15 July 1997     

Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri

Intercellular spreading of shigellae Is a prerequisite for shigellosis, although the molecular mechanisms underlying the phenomenon are still largely obscure. To elucidate some of these mechanisms, we performed random TniO insertion mutagenesis in Shigella flexneri YSH6000T and found a chromosomal locus in the Notl-J segment responsible for bacterial spreading. The locus affected in the mutant, designated vacJ, was neither involved in the invasion of epithelial cells nor in intracellular movement, but was required for intercellular spread. The vacJ mutant was capable of forming bacterium-containing membranous protrusions within the infected cell, but had diminished ability to move from the protrusions into the cytoplasm of the adjacent epithelial cells. Cloning and sequencing of the vacJ region Indicated that the vacJ gene encoded a 28.0 kDa protein possessing a signal peptide at the N-terminus, which contained the motif characteristic of lipoproteins. The analysis of the vacJ product indicated that VacJ was exposed on the bacterial surface. The vacJ gene was distributed among shigellae and enteroinvasive Escherichia coli, and the constructed vacJ mutants failed to spread intercellularly, indicating that vacJ is a chromosomal gene essential for the pathogenicity of shigeiiae.     

Sds22, a PP1 phosphatase regulatory subunit, regulates epithelial cell polarity and shape [Sds22 in epithelial morphology]

Background  

How epithelial cells adopt their particular polarised forms is poorly understood. In a screen for genes regulating epithelial morphology in Drosophila, we identified sds22, a conserved gene previously characterised in yeast.     

Neutrophil Gelatinase-associated Lipocalin, a Siderophore-binding Eukaryotic Protein

NGAL (neutrophil gelatinase-associated lipocalin) also known as lcn2 or siderochalin is constitutively expressed in myelocytes and stored in specific granules of neutrophils. It is highly induced in a variety of epithelial cells during inflammation. Analysis of the crystal structure of NGAL expressed in E.coli showed that NGAL has the ability to bind catecholate type siderophores and in this way prevent bacteria from acquisition of siderophore-bound iron. NGAL (or 24p3 as the highly homologous murine orthologue is named) knock out mice have a profound defect in defense against E.coli after intraperitoneal injection. This defect can be mimicked in wild-type mice by providing siderophore iron, which cannot be sequestered by NGAL, testifying to the specific role of NGAL as a siderophore binding protein in innate immunity. Megalin, a scavenger receptor functions as a receptor for NGAL and mediates uptake into endosomes, but other NGAL receptors are likely to exist.     

Morphology,cell-cell interactions,and migratory activity of IAR-2 epithelial cells transformed with the <Emphasis Type="Italic">RAS</Emphasis> oncogene: Contribution of cell adhesion protein E-Cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration.     

TGF-β induces the expression of SAP30L, a novel nuclear protein

Background  

We have previously set up an in vitro mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. In this model the fibroblast-induced epithelial cell differentiation from secretory crypt cells to absorptive enterocytes is mediated via transforming growth factor-β (TGF-β), the major inhibitory regulator of epithelial cell proliferation known to induce differentiation in intestinal epithelial cells. The aim of this study was to identify novel genes whose products would play a role in this TGF-β-induced differentiation.     

Mitogenicity and Adjuvanticity of a Marine Bacterium,Vibrio anguillarum,in Mice

The effects of whole cells of three different O serotypes of Vibrio anguillarum on the murine immune response were studied. The addition of different doses (1–100/ig/ml) of V. anguillarum cells, as well as Salmonella typhimurium lipopolysaccharide, markedly increased the incorporation of [³H] thymidine into in vitro cultured spleen cells of C57BL/6 mice. All three serotype strains of V. anguillarum were able to induce the mitogenic effect at 10 μg/ml and 100 μg/ml, but serotype I strains were more potent than the others. Since pretreatment of spleen cells with rabbit anti-mouse thymocyte antiserum did not affect the mitogenic activity of V. anguillarum, Vibrio cells may be a B-lymphocyte mitogen. When sheep or horse erythrocytes and Vibrio cells were injected intraperitoneally into ddY mice, Vibrio cells exhibited an enhancing effect on antibody response in vivo, regardless of the different serotypes. Vibrio cells, when injected intraperitoneally into mice before the antigen, markedly suppressed the antibody response. Several days after the injection of Vibrio cells, these mice showed an enhanced carbon clearance activity. Acid phosphatase activity in their peritoneal cells was also augmented, suggesting that Vibrio cells activated macrophages in the mice.     

Cytogenetic, morphologic and oncogene analysis of a cell line derived from a heterologous mixed mullerian tumor of the ovary

Summary A cell line was established from a mixed mullerian tumor of the ovary and designated LN1. Histopathologic analysis of the fresh tumor specimen demonstrated a highly aneuploid heterologous tumor comprised of undifferentiated mesodermal components with carcinomatous cells present as a smaller population. Long-term in vitro culture resulted in the establishment of a cell line that exhibits an epithelial-like morphology and expresses epithelial antigens cytokeratin, epithelial membrane antigen, and carcinoma antigen TAG-72. These cells also express mesenchymal intermediate filaments, vimentin, and desmin. Karyotypic analysis revealed a basic triploid pattern with multiple chromosomal abnormalities, most notably an isochromosome of the short arm of five present in three copies. Analysis of oncogene expression revealed that LN1 cells constitutively express mRNA for c-ras, c-erbB2, and p53. The expression of mRNA for cellular oncogenes correlated with the presence of corresponding oncoproteins, p21^H-ras, p21^K-ras, and p185^erbB2 and mutant p53 protein. In summary, coexpression of epithelial and mesenchymal antigens by LN1 cells lends support to the hypothesis that epithelial and mesenchymal elements comprising mixed mullerian tumors of the ovary are derived from a common stem cell precursor. Furthermore, this cell line represents a functional in vitro model to evaluate the biologic activities of these unusual and highly aggressive ovarian malignancies.