Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium

Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127–1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)–PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.

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Relative Levels of mRNA Encoding Enamel Proteins in Enamel Organ Epithelia and Odontoblasts

Journal of Dental Research ◽

10.1177/154405910308201209 ◽

2003 ◽

Vol 82 (12) ◽

pp. 982-986 ◽

Cited By ~ 59

Author(s):

T. Nagano ◽

S. Oida ◽

H. Ando ◽

K. Gomi ◽

T. Arai ◽

...

Keyword(s):

Tooth Enamel ◽

Structural Proteins ◽

Enamel Organ ◽

Maturation Stage ◽

Mrna Levels ◽

Rt Pcr ◽

Enamel Protein ◽

Enamel Proteins ◽

Enamel Layer

Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RT-PCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000-fold higher than in odontoblasts under maturation-stage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enamel-dentin junction.

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Differential androgen and estrogen substrates specificity in the mouse and primates type 12 17β-hydroxysteroid dehydrogenase

Journal of Endocrinology ◽

10.1677/joe-07-0144 ◽

2007 ◽

Vol 194 (2) ◽

pp. 449-455 ◽

Cited By ~ 32

Author(s):

Pierre-Gilles Blanchard ◽

Van Luu-The

Keyword(s):

Site Directed Mutagenesis ◽

Kidney Cells ◽

Cdna Fragment ◽

Steroidogenic Enzyme ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Mouse Sequence ◽

293 Cells ◽

Mouse Tissues ◽

Clitoral Gland

Recently, we have shown that human and monkey type 12 17β-hydroxysteroid dehydrogenases (17β-HSD12) are estrogen-specific enzymes catalyzing the transformation of estrone (E1) into estradiol (E2). To further characterize this novel steroidogenic enzyme in an animal model, we have isolated a cDNA fragment encoding mouse 17β-HSD12 and characterized its enzymatic activity. Using human embryonic kidney cells (HEK)-293 cells stably expressing mouse 17β-HSD12, we found that in contrast with the human and monkey enzymes, which are specific for the transformation of E1 to E2, mouse 17β-HSD12 also catalyzes the transformation of 4-androstenedione into testosterone (T), dehydroepiandroster-one (DHEA) into 5-androstene-3β,17β-diol (5-diol), as well as androsterone into 5α-androstane-3α,17β-diol (3α-diol). Previously, we have shown that the specificity of human and monkey 17β-HSD12s for C18-steroid is due to the presence of a bulky phenylalanine (F) at position 234 creating steric hindrance, preventing the entrance of C19-steroids into the active site. To determine whether the smaller size of the corresponding leucine (L) in the mouse sequence is responsible for the entrance of androgenic substrates, we performed site-directed mutagenesis to substitute Leu 234 for Phe in the mouse enzyme. In agreement with our hypothesis, the mutated enzyme has a highly reduced ability to metabolize androgens. mRNA quantification in several mouse tissues using real-time PCR shows that mouse 17β-HSD12 mRNA is highly expressed in the female clitoral gland, male preputial gland, as well as in retroperitoneal fat and adrenal of both sexes. The differential androgenic/estrogenic substrate specificity of type 12 17β-HSD in the mouse and primates seems to agree with the observation that androgen and estrogen in the mouse are provided almost exclusively by gonads, while in primates an important part of these steroid hormones are produced locally from adrenal precursors.

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Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1

Biochemical Journal ◽

10.1042/bj20081340 ◽

2009 ◽

Vol 418 (1) ◽

pp. 93-101 ◽

Cited By ~ 29

Author(s):

Bei You ◽

Guijun Yan ◽

Zhiling Zhang ◽

Lin Yan ◽

Jing Li ◽

...

Keyword(s):

Troponin I ◽

Molecular Conformation ◽

Phosphorylation Site ◽

Dominant Negative ◽

Binding Assays ◽

Interacting Protein ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

293 Cells

Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129 and Thr143. Substitution of Thr31 with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr51, Thr129 or Thr143 with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr31 is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr31. Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.

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Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption

Dentistry Journal ◽

10.3390/dj9010007 ◽

2021 ◽

Vol 9 (1) ◽

pp. 7

Author(s):

Yusuke Makino ◽

Kaoru Fujikawa ◽

Miwako Matsuki-Fukushima ◽

Satoshi Inoue ◽

Masanori Nakamura

Keyword(s):

Bacterial Infections ◽

Tooth Movement ◽

Tooth Eruption ◽

Intercellular Adhesion Molecule ◽

Enamel Organ ◽

Pcr Analysis ◽

Maturation Stage ◽

Oral Epithelium ◽

Rt Pcr ◽

Inflammatory Reactions

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

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LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

Biochemical Journal ◽

10.1042/bj3450673 ◽

2000 ◽

Vol 345 (3) ◽

pp. 673-680 ◽

Cited By ~ 90

Author(s):

Sean P. COLLINS ◽

Junewai L. REOMA ◽

David M. GAMM ◽

Michael D. UHLER

Keyword(s):

Protein Kinase ◽

Cellular Localization ◽

Fluorescent Protein ◽

Phosphorylation Site ◽

Multiple Cancer ◽

Dependent Protein Kinase ◽

Rnase Protection ◽

Camp Dependent Protein Kinase ◽

Increased Risk ◽

Dependent Protein

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

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Transforming growth factor-β1 expression is up-regulated in maturation-stage enamel organ and may induce ameloblast apoptosis

European Journal Of Oral Sciences ◽

10.1111/j.1600-0722.2009.00612.x ◽

2009 ◽

Vol 117 (2) ◽

pp. 105-112 ◽

Cited By ~ 27

Author(s):

Masahiro Tsuchiya ◽

Ramaswamy Sharma ◽

Coralee E. Tye ◽

Toshihiro Sugiyama ◽

John D. Bartlett

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

Enamel Organ ◽

Maturation Stage

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Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans

AJP Renal Physiology ◽

10.1152/ajprenal.1998.275.4.f550 ◽

1998 ◽

Vol 275 (4) ◽

pp. F550-F564 ◽

Cited By ~ 14

Author(s):

Eli J. Holtzman ◽

Sumit Kumar ◽

Carol A. Faaland ◽

Fern Warner ◽

Paul J. Logue ◽

...

Keyword(s):

Amino Acids ◽

Human Kidney ◽

Gene Organization ◽

Kidney Cells ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

C Elegans ◽

Transmembrane Segments ◽

Extracellular Loops ◽

Stimulation Of

We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegansK-Cl cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share ∼45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains 10 exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.

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Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm

Biochemical Journal ◽

10.1042/bj20070292 ◽

2007 ◽

Vol 406 (1) ◽

pp. 31-40 ◽

Cited By ~ 23

Author(s):

Nobuhito Ono ◽

Ingrid Van der Heijden ◽

George L. Scheffer ◽

Koen Van de Wetering ◽

Elizabeth Van Deemter ◽

...

Keyword(s):

Multidrug Resistance ◽

Kidney Cells ◽

Cell Fractionation ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Hek 293 Cells ◽

Drug Conjugates ◽

293 Cells ◽

Boar Sperm ◽

Alternatively Spliced

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.

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Distribution of Calcium and Phosphate in Cells of the Enamel Organ in the Rat Lower Incisor

Advances in Dental Research ◽

10.1177/08959374870010021201 ◽

1987 ◽

Vol 1 (2) ◽

pp. 236-244 ◽

Cited By ~ 12

Author(s):

T. Kawamoto ◽

M. Shimizu

Keyword(s):

The Other ◽

Whole Body ◽

Enamel Organ ◽

Maturation Stage ◽

Lower Incisor ◽

X Ray ◽

Other Hand ◽

Freeze Dried ◽

Significant Difference ◽

Cell Layers

The distribution of calcium and phosphate in the cells of the enamel organ of the rat lower incisors was investigated by autoradiography and energy-dispersive x-ray spectrometry (EDS). Radioactive calcium or phosphate was injected i.p. into seven-day-old rats of the Wistar strain. The animals were frozen 0.5, 1, and 10 min after injection, and embedded in 5% carboxymethyl cellulose. Sagittal sections of 10 μm thickness were made in which the lower incisor was included as a part of the whole-body section. For autoradiography, the sections were freeze-dried and placed in contact with dry thin films prepared from autoradiographic emulsion. For EDS, sections were mounted on carbon stubs, freeze-dried, coated with carbon, and examined by EDS in a SEM. 45Ca and 32P autoradiograms showed that the radioactivity was located over the papillary layer cells adjacent to the secretory stage ameloblasts and was much higher here than in the ameloblastic layer. On the other hand, there was no significant difference between the amount of radioactivity of these two cell layers in the maturation stage, although higher radioactivity was detectable in the maturation stage enamel than in the secretory stage enamel. Pronounced Ka x-ray peaks were obtained for P, S, Cl, and K originating from the cells of the papillary and ameloblastic layers in the secretory stage, but only very low peaks were obtained for Ca. On the other hand, in addition to these elements, remarkably high Ca and Fe peaks could be detected in the ameloblastic layer of the maturation stage.

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Cecropia pachystachya Leaves Present Potential to Be Used as New Ingredient for Antiaging Dermocosmetics

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/8263934 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9

Author(s):

Maria Fernanda Fernandes ◽

Jessica Leiras Mota Conegundes ◽

Nícolas de Castro Campos Pinto ◽

Luiz Gustavo de Oliveira ◽

Jair Adriano Kopke de Aguiar ◽

...

Keyword(s):

Biological Activities ◽

Thiobarbituric Acid ◽

Skin Aging ◽

Total Phenolic ◽

Leaf Extracts ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Glycation End Products ◽

First Time

Several biological activities have been reported for leaf extracts of Cecropia pachystachya species, including antioxidant and wound healing activities. This study aims to report, for the first time, the antiaging potential of the hydroethanolic (HE) and the ethanolic (EE) extracts obtained from the leaves of C. pachystachya using different in vitro assays. Both HE and EE presented relevant antioxidant capacity in different models, including phosphomolybdenum, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), carotene/linoleic acid bleaching, and thiobarbituric acid reactive substances (TBARS) assays. Their ability to prevent the production of advanced glycation end products (AGEs) was also evaluated, and both extracts showed important activity, especially HE. The extracts also stimulated the fibroblasts proliferation in vitro, specialized cells that produce several mediators which maintain the skin integrity and youthfulness. Cytotoxicity of the extracts was not observed for this lineage or HEK-293, human embryonic kidney cells widely used to evaluate cytotoxicity of chemical compounds. HE also exhibited the ability to inhibit the collagenase (metalloproteinase MMP-2) and elastase activities. The total phenolic and flavonoids contents were also determined. HPLC analysis revealed the presence of the flavonoids orientin and iso-orientin, which were quantified to be used as chemical markers. The results suggested that the extracts of C. pachystachya leaves present the potential to be used in dermocosmetic formulations to prevent the skin aging process, which attracts the attention of pharmaceutical companies and researchers interested in the development of novel ingredients likely to be used as active principles in antiaging products.

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