Endocytosis of cholera toxin by human enterocytes is developmentally regulated

Many secretory diarrheas including cholera are more prevalent and fulminant in young infants than in older children and adults. Cholera toxin (CT) elicits a cAMP-dependent chloride secretory response in intestinal epithelia, which accounts for the fundamental pathogenesis of this toxigenic diarrhea. We have previously reported that the action of this bacterial enterotoxin is excessive in immature enterocytes and under developmental regulation. In this study, we tested the hypothesis that enhanced endocytosis by immature human enterocytes may, in part, account for the excessive secretory response to CT noted in the immature intestine and that enterocyte endocytosis of CT is developmentally regulated. To test this hypothesis, we used specific inhibitors to define endocytic pathways in mature and immature cell lines. We showed that internalization of CT in adult enterocytes is less and occurs via the caveolae/raft-mediated pathway in contrast to an enhanced immature human enterocyte CT uptake that occurs via a clathrin pathway. We also present evidence that this clathrin pathway is developmentally regulated as demonstrated by its response to corticosteroids, a known maturation factor that causes a decreased CT endocytosis by this pathway.     

An embryonal carcinoma cell line as a model system to study developmentally regulated genes during myogenesis

We have established conditions to efficiently differentiate embryonic carcinoma stem cells of the line P19 into myogenic cells. As inducers for differentiation, a combination of embryoid body formation in conjunction with treatment with dimethyl sulfoxide and retinoic acid proved to be most efficient. Under these conditions we detected an accumulation of myosin- and actin-specific RNA. Also, large amounts of type IV collagen RNA were produced. Type IV collagen is a component of the muscle basement membrane. In analogy to the F-9 system, we found a drastic decrease in stable p53 mRNA under the differentiation conditions used.     

Expression of rat alpha 2-macroglobulin gene during pregnancy

Rat alpha 2-macroglobulin (alpha 2M) is a typical acute phase protein, the concentration of which in serum increases more than 100-fold after inflammation. It is also known that the protein increases during pregnant (and neonatal) stages. Using a specific cDNA probe, expression of the alpha 2M gene during pregnancy was studied at the mRNA level. During inflammation, the liver is almost the only organ producing alpha 2M, but during pregnancy the placenta and uterus were found to be major organs producing a large amount (70-80% of that of inflamed liver) of alpha 2M mRNA at days 12-15. The yolk sac, maternal liver and fetal (or neonatal) liver also produced a small but significant amount (5-20% of that of inflamed liver) of the mRNA. Southern blotting analysis showed that only one copy of the alpha 2M gene was present in a haploid rat genome. These results indicated that a single alpha 2M gene has the ability to respond to two completely-different physiological states.     

ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis.

ADAMTS-like 2 (ADAMTSL2), is a secreted protein resembling the ancillary domains of the ADAMTS proteases, but with distinct structural features. It has 7 thrombospondin type-1 repeats (TSRs), but an unusually long spacer module, which in both humans and mice, contains a novel insertion bearing six N-glycosylation sites. The ADAMTSL2 protein expressed in HEK293F and COS-1 cells, is a cell-surface and extracellular matrix binding glycoprotein, with N-linked carbohydrate constituting approximately 20% by mass. The 4.0 kb Adamtsl2 mRNA is found most abundantly in adult mouse liver, lung and spleen by northern blotting. During mouse embryogenesis, Adamtsl2 was expressed most strongly in the third week of gestation. Adamtsl2 mRNA was detected by in situ hybridization in developing skeletal muscle, liver, bronchial and arterial smooth muscle, skin, intervertebral disc, perichondrium, pancreas and spinal cord. Immunohistochemical localization of ADAMTSL2 protein was similar to mRNA expression. Detection of Adamtsl2 mRNA and protein in developing skeletal myotubes, but not undifferentiated myogenic precursors led us to investigate its regulation during in vitro myogenic differentiation. In C2C12 and 23A2 myogenic cells, but not in 23A2 cells rendered non-myogenic by expression of G12V:H-Ras (9A2 cells), differentiation induced by serum starvation triggered expression of Adamtsl2 mRNA, coordinately with Myog, a marker of muscle differentiation. Furthermore, activation of the key myogenic determinant MyoD in 10T1/2 fibroblasts also triggered expression of Adamtsl2 mRNA. Collectively, the data suggest that induction of Adamtsl2 mRNA is an integral feature of myogenesis.     

Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant （K220R） caused precocious differentiation of ceUs into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.     

H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis [published erratum appears in J Cell Biol 1992 Jul;118(1):213]

H36 is a 120,000-D membrane glycoprotein that is expressed during the differentiation of skeletal muscle. H36 cDNA clones were isolated from a lambda UniZapXR rat myotube cDNA library and sequenced. The deduced amino acid sequence demonstrates that H36 is a novel integrin alpha chain that shares extensive homology with other alpha integrins that includes: (a) the GFFKR sequence found in all alpha integrins; (b) a single membrane spanning region; (c) conservation of 18 of 22 cysteines; and (d) a protease cleavage site found in the non-I region integrin alpha chains. The cytoplasmic domain of H36 is unique and additional regions of nonhomology further indicate H36 is distinct from all other alpha chains. In keeping with current nomenclature we designate this alpha chain alpha 7. Northern blots demonstrate that expression of H36-alpha 7 mRNA is regulated both early in the development of the myogenic lineage and later, during terminal differentiation. Detection of H36-alpha 7 mRNA coincides with conversion of H36- myogenic precursor cells to H36+ cells. H36-alpha 7 mRNA is present in replicating myoblasts: expression increases upon terminal differentiation and is markedly reduced in developmentally defective myoblasts. In addition, H36-alpha 7 mRNA is not detected in C3H10T1/2 cells. It is in myotubes derived from myoblasts obtained by treatment of 10T1/2 cells with azacytidine or transfection with MRF4. Immunoblots and immunofluorescence demonstrate that the H36-alpha 7 chain is associated with integrin beta 1. Affinity chromatography demonstrates that H36-alpha 7 beta 1 selectively binds to laminin. The expression of H36-alpha 7 on secondary myoblasts during the development of the limb in vivo corresponds with the appearance of laminin in the limb, with the responsiveness of secondary myoblast proliferation to laminin, and with the onset of increased muscle mass, suggesting that H36-alpha 7 modulates this stage in limb development. We conclude that H36-alpha 7 is a novel alpha integrin laminin binding protein whose expression is developmentally regulated during skeletal myogenesis.     

Evolution of human alpha 2-macroglobulin

A papain-binding protein (PBP) resembling human alpha 2-macroglobulin (alpha 2M) but of Mr half that of alpha 2M was purified from plaice (Pleuronectes platessa L.) plasma. The plaice protein displayed most of the distinctive inhibitory properties of the human macroglobulin, and was therefore considered, despite its smaller molecular size, to be homologous with alpha 2M. Plaice PBP was shown to consist of four dissimilar subunits; two I chains (Mr 105 000) and two II chains (Mr 90 000). Each of the larger I chains contained a "bait region" sensitive to proteolytic attack by a variety of proteinases, and an autolytic site analogous to the autolytic site of alpha 2M. Subunit I, almost certainly at the autolytic site, formed SDS-stable, covalent links with methylamine or a proportion of the trapped proteinase molecules. A scheme is proposed for the evolution of human alpha 2M from the smaller fish protein, and the possibility of a shared evolutionary origin for alpha 2M and the complement components C3 and C4 is discussed.     

Antiproteinase activity of alpha 2-macroglobulin

Blood serum separation by the method of gel filtration on Sephadex G-200 with the subsequent immunochemical determination of the quantitative content of basic proteolysis inhibitors permitted isolating the alpha 2-macroglobulin fraction while alpha 1-antitrypsin and alpha 1-antichymotrypsin separation was a failure. The immunochemical analysis of the antienzymic activity of the isolated inhibitors showed that 32.3 +/- 3.5% of the introduced kallikrein, 18.7 +/- 0.6% of trypsin and 14.4 +/- 4.1% of chymotrypsin were bound in the zone of alpha 2-macroglobulin. The rest of antienzymic activity was localized in the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin. After a preliminary saturation of blood serum with trypsin in the amount equivalent to its antitryptic capacity (200 micrograms/ml) the ability of alpha 2-macroglobulin to bind kallikrein and chymotrypsin lowers considerably (by 69 and 72%, respectively). In the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin a decrease in the ability to bind kallikrein and chymotrypsin amounted to 44 and 12% respectively. Thus, alpha 2-macroglobulin being bound with trypsin looses considerably its ability to bind other enzymes.     

Double-strand break repair in plants is developmentally regulated

In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional beta-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 x 10(-8)) being only 30% of the recombination rate at 2 dpg (9.14 x 10(-8)). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.     

Endocytosis of FcgammaRI is regulated by two distinct signalling pathways

Aggregation by immune complexes of receptors specific for the Fc region of IgG results in their internalisation and disposal by trafficking to lysosomes. We show here that internalisation of FcgammaRI by IFN-gamma treated U937 cells following receptor aggregation by cross-linking antibodies requires the activation of two distinct signalling pathways. The pathways were functionally dissected in streptolysin-O-permeabilised cells by capitalising on their relative dependence on active GTP binding proteins. One pathway required the presence of GTP-gammaS or active betagamma subunits, the other did not. Use of inhibitors revealed that the betagamma-independent pathway required activation of PI 3-kinases and was PKC-independent In contrast, the betagamma-dependent pathway involved activation of phospholipase C-beta and PKC, but was PI 3-kinase-independent. Both these pathways were found to be active in intact cells and are likely to determine receptor trafficking following internalisation.     

Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor alpha403 heterodimers is developmentally regulated during myeloid differentiation

The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARalpha exhibiting dominant-negative activity (RARalpha403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 microM) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARalpha in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR-RARalpha403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA-triggered GALdbd-RARalpha hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARalpha403 heterodimers that we observed at these different stages of myeloid development.     

Desmosomal adhesiveness is developmentally regulated in the mouse embryo and modulated during trophectoderm migration

During embryonic development tissues remain malleable to participate in morphogenetic movements but on completion of morphogenesis they must acquire the toughness essential for independent adult life. Desmosomes are cell-cell junctions that maintain tissue integrity especially where resistance to mechanical stress is required. Desmosomes in adult tissues are termed hyper-adhesive because they adhere strongly and are experimentally resistant to extracellular calcium chelation. Wounding results in weakening of desmosomal adhesion to a calcium-dependent state, presumably to facilitate cell migration and wound closure. Since desmosomes appear early in mouse tissue development we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion, the opposite of what happens on wounding. We show that epidermal desmosomes are calcium-dependent until embryonic day 12 (E12) and become hyper-adhesive by E14. Similarly, trophectodermal desmosomes change from calcium-dependence to hyper-adhesiveness as blastocyst development proceeds from E3 to E4.5. In both, development of hyper-adhesion is accompanied by the appearance of a midline between the plasma membranes supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of desmosomal cadherins. By contrast, adherens junctions remain calcium-dependent throughout but tight junctions become calcium-independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα-/- mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. We demonstrate that modulation of desmosomal adhesion by PKC can regulate migration of trophectoderm. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.     

Proteinase inhibitor II is developmentally regulated in Nicotiana flowers

Proteinase inhibitors (PI) are expressed in several members of the Solanaceae. According to species, PI can be both developmentally and environmentally regulated. We investigated the presence and developmental regulation of PI in the flowers of Nicotiana plumbaginifolia. By trypsin affinity chromatography a small protein, Np-PI-II, was isolated. This protein had an N-terminus similar to a part of a Nicotiana alata PI-II, and an inhibitory activity towards trypsin. Inhibitory activity is expressed at very early stages of floral development. No inhibitor is present in the vegetative meristems. The inhibitory activity remains high throughout flower maturation and rapidly declines in the ovary after pollination. In the stigma of mature Nicotiana tabacum flowers trypsin inhibitory activity is detected extracellularly. Proteinase inhibitor activity is not a regular feature of the stigmas of several solanaceous genera.
Proteinase inhibitors apparently have no function in the development of floral structures and probably act as protective agents against fungal infection of and insect damage to the floral parts and the developing fruit.