Truncated amelogenin and LRAP transgenes improve Amelx null mouse enamel

Amelogenin is the most abundant enamel protein involved in enamel mineralization. Our goal was to determine whether all three regions of amelogenin (N-terminus, C-terminus, central core) are required for enamel formation. Amelogenin RNA is alternatively spliced, resulting in at least 16 different amelogenin isoforms in mice, with M180 and LRAP expressed most abundantly. Soon after secretion by ameloblasts, M180 is cleaved by MMP20 resulting in C-terminal truncated (CTRNC) amelogenin. We aimed to determine whether the 2 transgenes (Tg), LRAP and CTRNC together, can improve LRAPTg/Amelx −/− and CTRNCTg/Amelx −/− enamel thickness and prism organization, which were not rescued in Amelx −/− enamel. We generated CTRNCTg/LRAPTg/Amelx −/− mice and analyzed developing and mature incisor and molar enamel histologically, by microCT, SEM and microhardness testing. CTRNCTg and LRAPTg overexpression together significantly improved the enamel phenotype of LRAPTg/Amelx −/− and CTRNCTg/Amelx −/− mouse enamel, however enamel microhardness was recovered only when M180Tg was expressed, alone or with LRAPTg. We determined that both LRAP and CTRNC, which together express all three regions of the amelogenin protein (N-terminus, C-terminus and hydrophobic core) contribute to the final enamel thickness and prism organization in mice.     

A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein)

Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up > 90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the ¹H-¹⁵N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the ¹H-¹⁵N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of ¹H-¹⁵N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of ¹H-¹⁵N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein.     

Leucine-rich amelogenin peptide induces osteogenesis by activation of the Wnt pathway

We previously showed that one of the amelogenin splicing isoforms, Leucine-rich amelogenin peptide (LRAP), induced osteogenic differentiation of mouse embryonic stem cells; however, the signaling pathway(s) activated by LRAP remained unknown. Here, we demonstrated that the canonical Wnt/β-catenin signaling is activated upon LRAP treatment, as evidenced by elevated β-catenin level and increased Wnt reporter gene activity. Furthermore, a specific Wnt inhibitor sFRP-1 completely blocks the LRAP-mediated Wnt signaling. However, exogenous recombinant Wnt3a alone was less effective at osteogenic induction of mouse ES cells in comparison to LRAP. Using a quantitative real-time PCR array, we discovered that LRAP treatment up-regulated the expression of Wnt agonists and down-regulated the expression of Wnt antagonists. We conclude that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists.     

The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

Amelogenin is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein–surface interactions are critical to its function. We have previously used LRAP, a splice variant of amelogenin, as a model protein for the full-length amelogenin in solid-state NMR and neutron reflectivity studies at interfaces. In this work, we examined the adsorption behavior of LRAP in greater detail using model self-assembled monolayers containing COOH, CH₃, and NH₂ end groups as substrates. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline and solutions containing low concentrations of calcium and phosphate consisted of aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and quaternary structures on the surfaces. Relatively high amounts of adsorption occurred onto the CH₃ and NH₂ surfaces from both buffer solutions. Adsorption was also promoted onto COOH surfaces only when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies revealed that LRAP adsorbed onto the surfaces as small subnanosphere-sized structures such as monomers or dimers. We propose that the monomers/dimers were present in solution even though they were not detected by DLS or that they adsorbed onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces.     

Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells

Leucine-rich amelogenin peptide (LRAP), an alternatively spliced amelogenin protein, possesses a signaling property shown to induce osteogenic differentiation. In the current study, we detected LRAP expression during osteogenesis of wild-type (WT) embryonic stem (ES) cells and observed the absence of LRAP expression in amelogenin-null (KO) ES cells. We explored the signaling effect of LRAP on wild-type ES cells, and the ability of LRAP to rescue the impaired osteogenesis phenotype observed in KO ES cells. Our data indicate that LRAP treatment of WT and KO ES cells induces a significant increase in mineral matrix formation, and significant increases in bone sialoprotein and osterix gene expression. In addition, the amelogenin KO phenotype is partially rescued by the addition of exogenous LRAP. These data suggest a unique function of LRAP during ES cell differentiation along osteogenic lineage.     

Hypoxia-inducible factor 1-alpha acts as a bridge factor for crosstalk between ERK1/2 and caspases in hypoxia-induced apoptosis of cementoblasts

Hypoxia-induced apoptosis of cementoblasts (OCCM-30) may be harmful to orthodontic treatment. Hypoxia-inducible factor 1-alpha (HIF-1α) mediates the biological effects during hypoxia. Little is known about the survival mechanism capable to counteract cementoblast apoptosis. We aimed to investigate the potential roles of HIF-1α, as well as the protein-protein interactions with ERK1/2, using an in-vitro model of chemical-mimicked hypoxia and adipokines. Here, OCCM-30 were co-stimulated with resistin, visfatin or ghrelin under CoCl₂-mimicked hypoxia. In-vitro investigations revealed that CoCl₂-induced hypoxia triggered activation of caspases, resulting in apoptosis dysfunction in cementoblasts. Resistin, visfatin and ghrelin promoted the phosphorylated ERK1/2 expression in OCCM-30 cells. Furthermore, these adipokines inhibited hypoxia-induced apoptosis at different degrees. These effects were reversed by pre-treatment with ERK inhibitor (FR180204). In cells treated with FR180204, HIF-1α expression was inhibited despite the presence of three adipokines. Using dominant-negative mutants of HIF-1α, we found that siHIF-1α negatively regulated the caspase-8, caspase-9 and caspase-3 gene expression. We concluded that HIF-1α acts as a bridge factor in lengthy hypoxia-induced apoptosis in an ERK1/2-dependent pathway. Gene expressions of the caspases-3, caspase-8 and caspase-9 were shown to be differentially regulated by adipokines (resistin, visfatin and ghrelin). Our study, therefore, provides evidence for the role of ERK1/2 and HIF-1α in the apoptotic response of OCCM-30 cells exposed to CoCl₂-mimicked hypoxia, providing potential new possibilities for molecular intervention in obese patients undergoing orthodontic treatment.     

Dental enamel matrix: Sequences of two amelogenin polypeptides

The amino acid sequences of a leucine-rich amelogenin polypeptide (LRAP) and a tyrosine-rich amelogenin polypeptide (TRAP), isolated from foetal bovine enamel matrix, were determined. Both LRAP and TRAP occurred in two forms; in each case, one of the molecular species appeared to be shortened at the COOH terminus by 2 and 4 residues, respectively. A striking finding was that LRAP and TRAP had identical sequences for the first 33 residues but were almost completely different for the remaining 12 amino acids.     

The effects of LAMP1 and LAMP3 on M180 amelogenin uptake, localization and amelogenin mRNA induction by amelogenin protein

We previously demonstrated that the uptake of M180 amelogenin protein in dental epithelial cells (HAT-7) results in increased levels of amelogenin mRNA through enhanced mRNA stabilization. To determine the processes involved in the uptake of extracellular M180 amelogenin by cells and in amelogenin intracellular trafficking in the amelogenin protein-mediated amelogenin mRNA expression pathway, we investigated the effects of LAMP1 and LAMP3, which are candidate M180 amelogenin receptors, on M180 amelogenin uptake, localization and amelogenin mRNA induction by amelogenin protein, using anti-LAMP-1 and anti-LAMP-3 antibodies and siRNA analysis. The results indicate that LAMP3 blocking by anti-LAMP-3 decreases M180 amelogenin uptake, but does not affect amelogenin mRNA induction by amelogenin protein, suggesting that LAMP3 is related to amelogenin degradation. Down-regulation by siRNA of LAMP1, which is the receptor for small amelogenin protein (LRAP), does not affect M180 amelogenin uptake, localization or amelogenin mRNA induction by amelogenin protein. Thus, while LAMP1 is the specific receptor for LRAP, it is not a receptor for M180 amelogenin. These findings will aid further research into the understanding of M180 amelogenin function and expression.     

A reliable, non-invasive PCR method for giant panda (Ailuropoda melanoleuca) sex identification

We developed an inexpensive, fast and reliable PCR method for sex identification of giant panda (Ailuropoda melanoleuca) by using one pair of primers to co-amplify homologous fragments with size polymorphism that located at amelogenin (AMEL) exon 5. In giant panda, a 63 bp deletion in exon 5 of Y-linked allele provides a significant discrimination between AMELX and AMELY, thus the amplification products can be distinguished simply by agarose gel electrophoresis, exhibiting sex-specific banding patterns (male: 237 bp, 174 bp; female: 237 bp). Both blood and feces samples from known-sex giant pandas were successfully amplified. Cross species test also revealed that this method could be applied to other Ursidae species. These authors contributed equally to this work.     

Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

Background  

Expression of the urokinase plasminogen activator receptor (UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC), airway smooth muscle (HASM) and peripheral cells).     

Sulfated Glycosaminoglycans Enhance Tumor Cell Invasionin Vitroby Stimulating Plasminogen Activation

Metastasizing tumor cells invade host tissues by degrading extracellular matrix constituents. We report here that the highly sulfated glycosaminoglycans, heparin and heparan sulfate, as well as the sulfated polysaccharide, fucoidan, significantly enhanced tumor cell invasionin vitrointo fibrin, the basement membrane extract, Matrigel, or through a basement membrane-like extracellular matrix. The enhancement of tumor cell invasion was due to a stimulation of the proteolytic cascade of plasminogen activation since the effect required plasminogen activation and was abolished by inhibitors of urokinase-type plasminogen activator (uPA) or plasmin. Sulfated polysaccharides enhanced five reactions of tumor-cell initiated plasminogen activation in a dose-dependent manner. They amplified plasminogen activation in culture supernatants up to 70-fold by stimulating (i) pro-uPA activation by plasmin and (ii) plasminogen activation by uPA. (iii) In addition, sulfated polysaccharides partially protected plasmin from inactivation by α₂-antiplasmin. Sulfated polysaccharides also stimulated tumor-cell associated plasminogen activation, e.g., (iv) cell surface pro-uPA activation by plasmin and (v) plasminogen activation by cell surface uPA. These results suggest that sulfated glycosaminoglycans liberated by tumor-cell mediated extracellular matrix degradationin vivomight amplify pericellular plasminogen activation and locally enhance tumor cell invasion in a positive feedback manner.     

Ammonium assimilation in root nodules of actinorhizal Discaria trinervis. Regulation of enzyme activities and protein levels by the availability of macronutrients (N,P and C)

Asparagine was found to be the main N compound exported from Discaria trinervis nodules. Aspartate (Asp), glutamate (Glu), alanine (Ala) and serine (Ser) were also detected in root xylem sap, but at lower concentrations. A comparable picture is found in nodulated alfalfa. We hypothesized that a similar set of enzymes for Asn synthesis was present in D. trinervis nodules. We demonstrate the expression of most of the enzymes involved in the synthesis of Asn from NH⁺ ₄ and oxoacids, in nodules – but not in roots – of fully symbiotic D. trinervis. By complementation of enzyme assays (^A) and immunodetection (^I) we detected glutamane-synthetase (GS^{A, I}), Asp-aminotransferase (AAT^A), malate-dehydrogenase (MDH^{A, I}, at least two isoforms), Glu-dehydrogenase (GDH^A), Glu-synthase (GOGAT^I) and Asn-synthetase (AS^I). PEP-carboxylase (PEPC) activity was not detected. We previously shown that N acts as a negative regulator of nodulation and nodule growth, while P is a strong stimulator for nodule growth. We present data on the regulation of nodule N metabolism by altering, during 4 weeks, the availability of N, P and light in symbiotic D. trinervis. NH₄NO₃ (2 mM) induced inactivation and degradation of nodule GS, MDH and AS, but activation of GDH and AAT; the amount of nitrogenase components was not affected. A 10-fold increase in P supply did not greatly affect activity and amount of enzymes, suggesting that N metabolism is not P-limited in nodules. On the other hand, suppression of P supply induced an important reduction of nodule GS, GOGAT, MDH and AS protein levels, although nitrogenase was not affected. GDH was the only measured activity that was stimulated by limiting P supply. Shading plants did result in complete degradation of nitrogenase and partial degradation of GS, AS and nodule-specific MDH isoform, but GDH and AAT were activated. These results are discussed in connection with the regulation of nodulation and nodule growth in D. trinervis.     

Effect of metformin on ossification and inflammation of fibroblasts in ankylosing spondylitis: An in vitro study

Ankylosing spondylitis (AS) is an autoimmune disease characterized by fibroblasts ossification. However, effective drug therapy for AS is lacking. As an antidiabetic drug, metformin has demonstrated an antiosteogenic effect on osteoblasts in vitro. And it is also a kind of specific agonists for adenosine 5′‐monophosphate activated protein kinase (AMPK), which is blocked in the process of AS. Given the role in antiosteogenesis and AMPK activating, metformin was investigated of its effect on fibroblasts harvested from capsular ligament of patients with femoral neck fracture and AS. Osteogenic specific makers (Alp, Bglap, Runx2, Bmp2, and Col1) in fibroblasts administered with metformin (20 μg/mL) were detected by ALP staining, alizarin red staining, qPCR, and Western blotting after 7 and 14 days of culture. Inflammation genes (il1‐β and il6) and pathway (Pi3k, Akt, and Ampk) associated markers were also evaluated. Our results showed that osteogenic specific markers were greatly downregulated and ossification was effectively inhibited in AS fibroblasts after addition of metformin. Levels of inflammation markers were also decreased by metformin. Thus, metformin exerts potent effect on suppression of ossification and inflammation in AS fibroblasts via the activation of Pi3k/Akt and AMPK pathways, which may be developed as a potential agent for treatment of AS.     

Amelogenin gene similarity in vertebrates: DNA sequences encoding amelogenin seem to be conserved during evolution

Summary Mouse amelogenin cDNA was used in hybridization assays with genomic DNA, cut with the restriction enzyme Eco RI, from the edentulous chicken (Gallus domesticus), the monophyodont mouse (as control), diphyodont man, and the polyphyodont fishes Atlantic salmon (Salmo salar) and seawolf (Anarrhichas lupus). The hybridization assay was performed under stringent conditions with non-radioactive probes. Hybridization was obtained with mouse (6.4-kb band), man (9-kb and 13-kb bands), and seawolf (18-kb band) genomic DNA. This demonstrates DNA sequence similarities between these species, and supports the theory that DNA sequences encoding enamel proteins appear to be highly conserved during the evolution of vertebrates. Lack of hybridization in salmon and chicken may be due to sequence divergencies or structural differences in an amelogenin gene analog, or it may be that no amelogenin gene is present in these animals.     

Über die käfer- und spinnenfauna eines nordwestdeutschen Birkenbruchs

Zusammenfassung In einem Birkenbruch (Betuletum pubescentis) des nordwestdeutschen Flachlands wurde der Artenbestand an Spinnen (Araneae), Laufkäfern (Col. Carabidae) und Kurzflügelkäfern (Col. Staphylinidae) aufgenommen. Die Verteilung der Arten auf die Strauchschicht, die Krautschicht, das Torfmoos und die Streuschicht wird in Tabellen dargestellt. Die Ökologie der bezeichnenderen Arten wird kurz charakterisiert.

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Summary In the lowland of North-western Germany the animal community of a birch wood on peat-soil was investigated. This paper informs about the spiders (Araneae) and beetles (Col. Carabidae and Staphylinidae) found in the shrub layer, in the field layer, in the peat-moss, and in the surface litter. The ecology of some characteristic species is discussed.

     

<Emphasis Type="Italic">Petunia</Emphasis> × <Emphasis Type="Italic">hybrida</Emphasis> during transition to flowering as affected by light intensity and quality treatments

Petunia × hybrida was grown under high (H), medium (M) and low (L) light intensity [photoperiod; 16 h d⁻¹, photosynthetic photon flux density (PPFD); 360, 120 and 40 μmol m⁻² s⁻¹, respectively] as well as under end-of-day (EOD) red (R) and far-red (FR) light quality treatments [photoperiod; 14.5 h d⁻¹, PPFD; 30 μmol m⁻² s⁻¹ EOD; 15 min, Control (C) light; without EOD light treatment]. Shoot growth, leaf anatomical and photosynthetic responses as well as the responses of peroxidase (POD) isoforms and their specific activities following transition to flowering (1–6 weeks) were evaluated. Flower bud formation of Petunia × hybrida was achieved at the end of the 4th week for H light treatment and on the end of the 6th week for FR light treatment. No flower bud formation was noticed in the C and R light treatments. H and M light treatments induced lower chlorophyll (Chla, Chlb, Chla+b) concentrations in comparison to L light. On the other hand R and FR light chlorophyll content were similar to C light. Photosynthetic parameters [CO₂ assimilation rate (A), transpiration rate (E) and stomatal conductance (g _s) values] were higher in the H light treated plants in comparison to M and L light treated plants. A, E and g _s values of R and FR light were similar to C light plants. Leaf anatomy revealed that total leaf thickness, thickness of the contained tissues (epidermis, palisade and spongy parenchyma) and relative volume percentages of the leaf histological components were differently affected within the light intensity and the light quality treatments. POD specific activities increased from the 1st to the 6th week during transition to flowering. Native-PAGE analysis revealed the appearance of four anionic POD (A₁–A₄) isoforms in all light treatments. On the basis of the leaf anatomical, photosynthetic and plant morphological responses, the production of high quality Petunia × hybrida plants with optimal flowering times could be achieved through the control of both light intensity and light quality. The appearance of A₁ and A₂ anionic POD isoforms could be also used for successful scheduling under light treatments.