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PubMed1986

Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum.

UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition.

Cell Aggregation↗
PubMed1990

Nerve growth factor expression in the developing hippocampus isolated in vitro.

Nerve growth factor (NGF) is synthesized in the hippocampus and neocortex and provides trophic support for afferent cholinergic neurons of the basal forebrain. To determine the capacity of the developing hippocampus to express NGF in the absence of NGF-responsive afferents, embryonic hippocampal cells isolated prior to septal innervation were studied in reaggregating cell culture. The expression of NGF protein in vitro was qualitatively and quantitatively similar to that observed in situ. The expression of NGF mRNA exhibited an initial increase in vitro but then plateaued and was maintained at a steady level. This latter finding was in contrast to the steady rise in NGF mRNA levels observed in situ. These data suggest that (i) intrinsic hippocampal interactions regulate the onset of NGF expression, but that (ii) additional extrinsic developmental signals may be required for proper regulation of hippocampal NGF expression during ontogeny.

Aging↗
PubMed1992

Transient expression of syndecan in mesenchymal cell aggregates of the embryonic kidney.

Induction of the embryonic kidney mesenchyme is followed by formation of cell aggregates which subsequently transform into epithelial tubules. Syndecan, which binds various matrix components and growth factors, is a candidate molecule to be involved in this process. We have analyzed the changes in the expression of syndecan during tubule morphogenesis by using in situ hybridization and slot-blot analysis. The expression pattern of syndecan was compared with the distribution of cell proliferation analyzed by immunohistochemistry. Furthermore, the expression of syndecan during formation of the pretubular aggregates was studied in hanging-drop cultures of experimentally induced mesenchymal cells. Syndecan mRNA was expressed in the metanephric mesenchyme prior to induction, was intensely present during formation of the pretubular cell aggregates, but was lost during maturation of the nephron. Slot-blot analyses of the kidney mesenchymes (11-day kidney) cultured in a transfilter situation with a heterotypic inductor tissue that triggers a complete tubulogenic program in the nephric mesenchyme during the first 24 hr suggested the presence of syndecan mRNA in the uninduced mesenchymes with no change during induction. Expression of mRNA was stimulated later (13-day kidney) followed by subsequent decrease. Immunoisolation of sulfate-labeled syndecan, however, revealed a marked stimulation in the induced kidney mesenchyme during the first 24-hr inductive period when the DNA level still remained constant. In hanging-drop cultures where either induced or uninduced mesenchymal cells were dissociated and reaggregated, syndecan was detected only in the induced and aggregating mesenchymal cells. Double-immunostaining demonstrated a close correlation between syndecan expression and cell proliferation analyzed by bromodeoxyuridine incorporation. Thus, it appears that syndecan expression in the mesenchyme is initially induced post-transcriptionally and later during differentiation at the mRNA level. Syndecan may have a dual function during early kidney morphogenesis; it may be involved in cell aggregation through its adhesive properties, and it may contribute to proliferation of the induced mesenchymal cells by binding growth factors.

Animals↗
PubMed1975

Mutant of Dictyostelium discoideum defective in cell contact regulation of enzyme expression.

Previous work has shown that aggregation and disaggregation of cells during development of D. discoldeum significantly affects the expression of certain developmentally regulated enzymes. We have examined this cell contact regulation in a previously isolated mutant, Fr-17, and found that during the course of its developmental sequence it becomes specifically defective in this function.

Adenosine↗
PubMed1988

Electron microscopy of nuclear matrices prepared in situ under oxidizing conditions.

Nuclear matrices (NM) were prepared from mouse 3T3 fibroblasts attached to growth support film in the continuous presence of the disulfide cross-linking reagent, sodium tetrathionate. Intact cells and samples at each stage of NM preparation were fixed, embedded in Epon-Araldite, sectioned and stained conventionally with uranyl-lead. Nuclear size and shape changed little during extraction, but nuclei showed a gradual reduction in internal fibrogranular elements up to 1M NaCl, after which larger spaces were visible in the nucleoplasm. In contrast to similar samples prepared under reducing conditions in other studies, final NMs contained a highly ramified internal network of fibers and granular aggregates.

Animals↗
PubMed2002

Temporal expression of CD44 during embryonic chick limb development and modulation of its expression with retinoic acid.

Hyaluronan-cell interactions are initiated co-ordinately with mesenchymal condensation during chondrogenic differentiation in the limb bud. Hyaluronan is responsible for the retention and organization of proteoglycan within the cartilage matrix. Hyaluronan-CD44 binding also retains proteoglycan aggregates to the chondrocyte plasma membrane. A sequence for CD44 protein in chick has recently been reported, but never evaluated in chick chondrocytes. Total RNA was isolated from embryonic chick limb buds, stages 18, 19, 24, 25 and 30. Using semi-quantitative RT-PCR, expression of aggrecan, this chick CD44 orthologue and GAPDH mRNA was analyzed. Aggrecan expression was detected at all stages, but was increased at stage 30. CD44 mRNA was detected at extremely low levels at stage 18 to higher levels in the latter stages. Thus, the temporal expression of CD44 mRNA correlated with the onset of pre-cartilage condensation. The full-length chick chondrocyte CD44 cDNA was obtained following RT-PCR using RNA derived from tibial chondrocytes from stage 37 chick embryos. The nucleotide sequence was used to generate an amino acid sequence and analyses revealed homologies of 44.4% with mouse, 47.8% with bovine and 46.3% with human CD44. Tibial chondrocytes were cultured in the presence or absence of retinoic acid for 36 or 72 h. By RT-PCR, expression of aggrecan and the CD44 mRNA by chick chondrocytes was decreased after retinoic acid treatment, while GAPDH expression showed no change. As expected, control chondrocytes exhibited a round morphology while retinoic acid-treated chondrocytes were elongated. The retinoic acid-treated chondrocytes also exhibited reduced hyaluronan binding. This functional assay indicates a role for a CD44 receptor in matrix retention by chick chondrocytes.

Amino Acid Sequence↗
PubMed2006

Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase.

Mer (MerTK) is a receptor tyrosine kinase important in platelet aggregation, as well as macrophage cytokine secretion and clearance of apoptotic cells. Mer is not normally expressed in thymocytes or lymphocytes; however, ectopic Mer RNA transcript and protein expression is found in a subset of acute lymphoblastic leukemia cell lines and patient samples, suggesting a role in leukemogenesis. To investigate the oncogenic potential of Mer in vivo, we created a transgenic mouse line (Mer(Tg)) that expresses Mer in the hematopoietic lineage under control of the Vav promoter. Ectopic expression and activation of the transgenic Mer protein was demonstrated in lymphocytes and thymocytes of the Mer(Tg) mice. At 12-24 months of age, greater than 55% of the Mer(Tg) mice, compared to 12% of the wild type, developed adenopathy, hepatosplenomegaly, and circulating lymphoblasts. Histopathological analysis and flow cytometry were consistent with T-cell lymphoblastic leukemia/lymphoma. Mer may contribute to leukemogenesis by activation of Akt and ERK1/2 anti-apoptotic signals, which were upregulated in Mer(Tg) mice. Additionally, a significant survival advantage was noted in Mer(Tg) lymphocytes compared to wild-type lymphocytes after dexamethasone treatment. These data suggest that Mer plays a cooperative role in leukemogenesis and may be an effective target for biologically based leukemia/lymphoma therapy.

Animals↗
PubMed1995

Comparison of the Dictyostelium rasD and ecmA genes reveals two distinct mechanisms whereby an mRNA may become enriched in prestalk cells.

The Dictyostelium ras gene, rasD, encodes an mRNA that is more abundant in prestalk than prespore cells in the migratory slug. Its expression is inducible by extracellular cAMP but is not inducible by the prestalk and stalk cell morphogen differentiation inducing factor (DIF). We show that a rasD-lacZ fusion gene is first expressed in approximately one half of the cells in the aggregate, including some cells that also express a prespore-specific marker. The amount of rasD-lacZ fusion protein in prespore cells then diminishes as the slug is formed. Analysis of a rasD-lacZ fusion protein with an N terminal substitution that reduces protein stability within the cell provides strong confirmatory evidence that the ras gene product becomes enriched in prestalk cells by selective repression of gene expression in prespore cells. In contrast, the DIF-inducible ecmA gene is expressed only in those cells that will become prestalk cells in the migratory slug. These results show that there are two different ways in which an mRNA may become enriched in prestalk cells and support the view that DIF is the inducer of prestalk cell differentiation.

Animals↗
PubMed2005

Platelet endothelial aggregation receptor 1 (PEAR1), a novel epidermal growth factor repeat-containing transmembrane receptor, participates in platelet contact-induced activation.

The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.

Amino Acid Sequence↗
PubMed1994

Lautropia mirabilis gen. nov., sp. nov., a gram-negative motile coccus with unusual morphology isolated from the human mouth.

An organism that seems to be identical to Orskov's 'Sarcina mirabilis' [Orskov, J. (1930) Acta Pathol Microbiol Scand Suppl III, 519-541] has been rediscovered in specimens from the upper respiratory tract of humans. Six strains were studied, and the results, which conformed to Orskov's description of S. mirabilis, were as follows. Rough to smooth colonies grow on many plated media and show extremely polymorphic cell morphology with round cells with diameters from 1 to > 10 microns. The smallest cells were often motile with circular movements. Strains were Gram-negative, facultatively anaerobic, oxidase and urease positive, and weakly catalase positive. Nitrate and nitrite were reduced, and glucose, fructose, sucrose and mannitol were fermented. Polysaccharide was produced on sucrose agar. Electron microscopy showed coccoid cells with a bundle of three to nine flagella, a Gram-negative cell-wall morphology, and aggregates of irregular cells held together by a common surface layer. The mean mol% (G+C) of the organisms was 65.0. 16S-ribosomal RNA sequencing revealed that the organism belongs to the beta subgroup of Proteobacteria, separate from all other described genera, but most closely related to Burkholderia. The name Lautropia mirabilis is proposed for this organism.

Bacterial Typing Techniques↗
PubMed1994

Induction of intercellular adhesion molecule-1 by lipopolysaccharide in canine alveolar macrophages.

Previous studies have demonstrated that alveolar macrophages (AMphis) adherent to plastic release interleukin-8 in response to lipopolysaccharide (LPS). We sought to determine whether LPS could also alter surface adhesion molecule expression and thus modulate additional AMphi adhesive interactions. Canine AMphis obtained by bronchoalveolar lavage of excised lung were adhered onto tissue culture plastic and exposed to LPS (0.01 to 100 ng/ml). Expression of beta 2 integrins and intercellular adhesion molecule-1 (ICAM-1) was subsequently determined by flow cytometry, a cDNA probe to canine ICAM-1 was used to quantify ICAM-1 mRNA, and changes in adhesion molecule function were assessed by evaluating the extent of homotypic aggregation. ICAM-1 and CD11a/CD18 were present on freshly isolated AMphis. CD11b/CD18 and CD11c/CD18 were expressed at lower levels. Nonadherent AMphis expressed a pattern of beta 2 integrin and ICAM-1 comparable to adherent cells. During short-term LPS stimulation (3 h), adherent AMphis increased both the synthesis and expression of ICAM-1. CD18 expression was either decreased or remained unchanged with LPS stimulation. LPS stimulation in vitro (> 0.01 ng/ml) enhanced the homotypic aggregation of adherent AMphis. Aggregation was blocked by monoclonal antibodies to ICAM-1 (CL18/6) and CD11a (R7.1) and CD18 (R15.7). Similar kinetics were found for expression of ICAM-1 and homotypic aggregation, suggesting that up-regulation of ICAM-1 is a major determinant of the LPS-stimulated aggregation of AMphis.

Animals↗
PubMed1994

Spatiotemporal profile of N-cadherin expression in the developing limb mesenchyme.

During embryonic limb development, chondrogenesis of the mesenchyme is preceded by a crucial cellular condensation phase. Because condensation is likely to result from specific cell-cell interactions, we have examined the possible involvement of N-cadherin, a Ca(2+)-dependent cell adhesion molecule, in this condensation event. Previously, we have reported that N-cadherin is expressed in chick embryonic limb bud, and that perturbation of N-cadherin-mediated cell adhesion significantly inhibits limb mesenchymal cellular aggregation and chondrogenesis both in vivo and in vitro. To further examine the relationship between N-cadherin expression and chondrogenesis, we have quantified and characterized the spatiotemporal expression of N-cadherin mRNA and protein in the developing chick embryonic limb, and analyzed the mechanism whereby exogenous Ca2+ stimulates chondrogenesis in vitro. Immunohistochemistry revealed that N-cadherin is expressed on mesenchymal cell surface in a developmentally specific manner during limb bud maturation. N-Cadherin was detected in the mesenchyme of the central core of the early limb bud, and was maximally expressed between stages 24 to 26, corresponding to the period of cellular condensation in vivo. Mature cartilage did not express N-cadherin. In chick limb mesenchyme micromass cultures in vitro, N-cadherin protein expression was seen associated with distinct cellular aggregates of condensing mesenchyme, but not in mature cartilaginous nodules or the mesenchyme situated between the condensing aggregates. In situ hybridization localized N-cadherin mRNA expression in condensing regions of limb mesenchyme in a stage 25/26 limb bud; at later stages, N-cadherin mRNA expression was seen in the perichondrium and the dense mesenchyme, but not in mature cartilage. The agreement between the mRNA and protein data thus suggests that N-cadherin expression in the developing limb bud is regulated at the transcriptional level. Western immunoblot analysis further confirmed that, from stages 19 to 36, the level of N-cadherin in the limb bud increased 5-fold through stage 25/26, then decreased during the progression of chondrogenesis and osteogenesis. A parallel profile was also seen in micromass limb mesenchyme cultures in vitro. The importance of N-cadherin for Ca2+ mediated mesenchymal cell aggregation and chondrogenesis was examined by selective, proteolytic dissociation of the mesenchymal cells with retention or removal of N-cadherin. Cells with N-cadherin were shown to exhibit Ca(2+)-dependent aggregation and were consistently more chondrogenic than those without N-cadherin. These results provide strong support for the functional role of N-cadherin in chondrogenesis.

Animals↗
PubMedDate not supplied

Biological activity of novel macrocyclic alkaloids (budmunchiamines) from Albizia amara detected on the basis of interaction with DNA.

Extracts derived from Albizia amara were found to demonstrate activity in a recently developed hplc system designed to detect compounds capable of interacting with DNA. Further investigation led to the procurement of four sets of alkaloid isolates X1-X4 that were found to be macrocyclic pithecolobine alkaloids. All four isolates interacted with calf thymus DNA and were generally cytotoxic with a battery of cultured mammalian cells. As determined with Salmonella typhimurium strain TM677, isolates X1 and X3 were bactericidal, but not mutagenic. Isolate X1 was found to inhibit the catalytic activity of DNA polymerase, RNA polymerase, and HIV-1 reverse transcriptase. With DNA polymerase, the reaction was shown to be inhibited in a manner that was competitive with respect to DNA. In addition, isolate X1 inhibited each of the following: platelet aggregation, human lymphocyte transformation, phorbol-ester-induced chemiluminescence with human granulocytes, and cyclooxygenase activity. Detection of these alkaloids on the basis of their interaction with DNA exemplifies the validity of this approach.

Alkaloids↗
PubMed1994

Effects of irradiation on cholinergic neurons and nerve growth factor mRNA in mouse foetal brain aggregation cultures.

Neurochemical and morphological markers were used to study the dose-response relationship and effects of different doses (0.5, 1.0 and 2.0 Gy) of X-irradiation on foetal brain cells in reaggregating cell cultures. Cells, prepared from mouse forebrain were irradiated on culture day 2, corresponding to embryonic day 16 in vivo. The cell reaggregates were monitored continually up to day 40 in vitro. The level of a neuronal marker for cholinergic neurons, namely acetylcholine esterase (AChE), increased linearly with the dose of irradiation after 10 days in vitro. Furthermore, by day 30 the activity of AChE had decreased to a level below that found in the control aggregates. In the 2-Gy treatment the level of choline acetyltransferase (ChAT), another cholinergic-neuron marker, increased during the first 20-30 days in culture but had declined to control levels by day 40. Using Northern blot analysis of total RNA prepared from these cell reaggregates, we determined relative changes in the level of expression of mouse nerve growth factor (NGF) mRNA subsequent to irradiation after 10 and 30 days in culture. The ratio between the level of expression of NGF and that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated and it was found that by day 10 in vitro the level of NGF had increased compared with control levels, in a dose-related manner. After 30 days in culture, the level of NGF mRNA was still high after the low-dose (0.5 Gy) treatment, whereas it had decreased below control levels after the higher-dose (1.0 and 2.0 Gy) treatment. The aggregates showed a tendency to deform and fuse together after irradiation: furthermore, the number of aggregates occurring as two or more grown together, increased with the X-ray dose. Aggregate size decreased with time in vitro and with irradiation dose. Thus, we showed that cholinergic and morphological markers are affected by irradiation and the alteration in AChE activity induced by irradiation correlated well with the changes in NGF mRNA.

Animals↗