Initial enamel crystals are not spatially associated with mineralized dentine

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adiacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.     

Functional and molecular insights into KSRP function in mRNA decay

KSRP is a single strand nucleic acid binding protein that controls gene expression at multiple levels. In this review we focus on the recent molecular, cellular, and structural insights into the mRNA decay promoting function of KSRP. We discuss also some aspects of KSRP-dependent microRNA maturation from precursors that are related to its mRNA destabilizing function. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Mitochondrial protein synthesis: Figuring the fundamentals,complexities and complications,of mammalian mitochondrial translation

Mitochondrial protein synthesis is essential for all mammals, being responsible for providing key components of the oxidative phosphorylation complexes. Although only thirteen different polypeptides are made, the molecular details of this deceptively simple process remain incomplete. Central to this process is a non-canonical ribosome, the mitoribosome, which has evolved to address its unique mandate. In this review, we integrate the current understanding of the molecular aspects of mitochondrial translation with recent advances in structural biology. We identify numerous key questions that we will need to answer if we are to increase our knowledge of the molecular mechanisms underlying mitochondrial protein synthesis.     

Multiphoton imaging of the dentine‐enamel junction

Multiphoton microscopy has been used to reveal structural details of dentine and enamel at the dentin‐enamel junction (DEJ) based on their 2‐photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). In dentine tubule 2PEF intensity varies due to protein content variation. Intertubular dentin produces both SHG and 2PEF signals. Tubules are surrounded by a thin circular zone with a lower SHG signal than the bulk dentine and the presence of collagen fibers perpendicular to the tubule longitudinal axis is indicated by strong SHG responses. The DEJ appears as a low intensity line on the 2PEF images and this was never previously reported. The SHG signal is completely absent for enamel and aprismatic enamel shows a homogeneous low 2PEF signal contrary to prismatic enamel. The SHG intensity of mantle dentine is increasing from the dentine‐enamel junction in the first 12 μm indicating a progressive presence of fibrillar collagen and corresponding to the more external part of mantle dentine where matrix metallo‐proteases accumulate. The high information content of multiphoton images confirms the huge potential of this method to investigate tooth structures in physiological and pathological conditions. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The microstructure of dentine in taxonomic and phylogenetic studies

Traditionally, physical anthropologists have focused their dental studies on features of teeth that can be observed with the unaided eye, measured with calipers, or observed with light microscopes. With the advent of the scanning electron microscope, there has been renewed interest in the use of the microstructure of enamel in phylogenetic reconstruction and taxonomic classification. The microstructure of dentine has received far less research attention than has enamel although several investigators have proposed that dentine has taxon specificity. We report the first results of a study in which we investigated the taxon specificity of dentine. In our study, we exposed the dentinal tubules on the mesial root surfaces of 12 Canis, 11 Papio, and 12 Homo mandibular first molars and compared tubule density and pattern among the three taxa. Based on these parameters, 91.43% of the teeth were correctly identified as being dog, baboon, or human, respectively. We conclude that dentine has taxon-specific structural characteristics, which can be used in anthropological investigations. To our knowledge, this is the first comparative study of the micro-structure of teeth utilizing statistical analysis.     

Sequencing of bovine enamelin ("tuftelin") a novel acidic enamel protein

Enamelins are a major group of 28-70-kDa acidic proteins rich in aspartic acid, glutamic acid, serine, and glycine found in developing and mature extracellular enamel; a unique and highly mineralized ectodermal tissue covering vertebrate teeth. They have been associated with the mineralization and structural organization of this tissue. In an attempt to elucidate the primary structure of enamelin, a 2674-base pair cDNA isolated from a bovine ameloblast-enriched, lambda Zap 2 expression library, was sequenced. The identity and localization of the deduced protein was confirmed by amino acid composition, enzyme-linked immunosorbent assay, Western blotting, indirect immunohistochemistry, and high resolution protein-A gold immunocytochemistry. The immunological techniques were employed using antibodies directed against synthetic peptides corresponding to the protein sequence deduced from the cloned cDNA sequence. The results reveal the deduced protein to be a novel acidic enamel protein. It contains 389 amino acids and has a calculated molecular weight of 43,814. Its amino acid composition is similar to that of "tuft" proteins (enamel matrix protein fragments remaining in the mature tissue). It contains one potential N-glycosylation site and 5 cysteine residues. Southern hybridization of the cloned cDNA with genomic bovine DNA indicated the existence of a single gene with one or more introns.     

蜥臀目霸王龙及有鳞目沧龙牙齿组织的微细结构

本文是利用扫描电子显微镜对陆栖恐龙tvrannosaurid与海栖渐增mosasaurid”牙齿结构进行的比较解剖学研究。化石采自加拿大RedDeerhiverValley上白里统Horse－shoeCanyon组。通过研究地层中出现的生物化石,特别是动物牙齿的组织结构,可以了解动物为了适应生活环境而发生的进化过程,也可以推测它们的系统发育关系。tyrannosaund与mosas。id都拥有锥状的同形齿,牙齿侧向扁平,且略向后弯曲。研究结果确认了tyrannosaurid的牙齿由于薄层的无柱釉质bPrismaticenamel）向齿质的侵人而造成许多的凹凸构造,此锯齿状构造沿着牙齿的前后缘,由牙齿的顶端分布至基部。因此tyrannosaurid的牙齿呈现着锐利的切缘;在这些凹凸状切缘的沟与小窝的深部可观察到有机物的沉积。但是类似的锯齿状构造只能在齿冠呈钝圆状的mosasaurid牙齿的基部附近观察到。我们以扫描电子显微镜（SEM）检索,确认两爬行类的齿质皆是属于中间型的真性齿质（intermediatetypeorthodentine）;所谓orthodentine即是细管齿质（tubulardentine）。tyrannosaurid的真性齿质的齿质小管只在齿质一釉质相接处Uentino—enamaljunction）附近放散出规则性的分歧与末枝。但mosasaurid的真性齿质的齿质J。管,在齿质的中间层与表层中,呈现着由复杂     

A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects

Ameloblastin (AMBN) is the second most abundant extracellular matrix protein produced by the epithelial cells called ameloblasts and is found mainly in forming dental enamel. Inactivation of its expression by gene knockout results in absence of the enamel layer and its replacement by a thin layer of dysplastic mineralized matrix. The objective of this study was to further characterize the enamel organ and mineralized matrix produced in the AMBN knockout mouse. However, in the course of our study, we unexpectedly found that this mouse is in fact a mutant that does not express the full-length protein but that produces a truncated form of AMBN. Mandibles from wild type and mutant mice were processed for morphological analyses and immunolabeling. Microdissected enamel organs and associated matrix were also prepared for molecular and biochemical analyses. In incisors from mutants, ameloblasts lost their polarized organization and the enamel organ detached from the tooth surface and became disorganized. A thin layer of dysplastic mineralized material was deposited onto dentin, and mineralized masses were present within the enamel organ. These mineralized materials generated lower backscattered electron contrast than normal enamel, and immunocytochemistry with colloidal gold revealed the presence of amelogenin, bone sialoprotein and osteopontin. In addition, the height of the alveolar bone was reduced, and the junctional epithelium lost its integrity. Immunochemical and RT–PCR results revealed that the altered enamel organ in the mutant mice produced a shorter AMBN protein that is translated from truncated RNA missing exons 5 and 6. These results indicate that absence of full-length protein and/or expression of an incomplete protein have direct/indirect effects beyond structuring of mineral during enamel formation, and highlight potential functional regions on the AMBN molecule.     

Structural analyses of the pre‐mRNA splicing machinery

Pre‐mRNA splicing is a critical event in the gene expression pathway of all eukaryotes. The splicing reaction is catalyzed by the spliceosome, a huge protein‐RNA complex that contains five snRNAs and hundreds of different protein factors. Understanding the structure of this large molecular machinery is critical for understanding its function. Although the highly dynamic nature of the spliceosome, in both composition and conformation, posed daunting challenges to structural studies, there has been significant recent progress on structural analyses of the splicing machinery, using electron microscopy, crystallography, and nuclear magnetic resonance. This review discusses key recent findings in the structural analyses of the spliceosome and its components and how these findings advance our understanding of the function of the splicing machinery.     

Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells evidence for a unique role in secretory-protein synthesis.

Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.     

A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIH1)

Amelogenesis imperfecta is characterized by the defective formation of tooth enamel. Here we present evidence that the X-linked form of this disorder (AIH1) is caused by a structural alteration in one of the predominant proteins in enamel, amelogenin. Southern blot analysis revealed a deletion extending over 5 kb of the amelogenin gene in males with the hypomineralization form of the AIH1. Carrier females were heterozygous for the molecular defect. The deletion appears to include at least two exons of the amelogenin gene and the extent of the deletion was verified by PCR analysis. The mutation was shown to segregate with the disease among 15 analyzed individuals belonging to the same kindred. Our results link a defect in the amelogenin gene to the abnormal formation of enamel. We thus conclude that the amelogenin protein has a role in biomineralization of tooth enamel.     

Amelogenesis in vitro: a model for studies of epithelial postsecretory processing during tissue-specific extracellular matrix biomineralization

The extracellular matrix (ECM) of developing mammalian enamel comprises a complex of unusual epithelial-derived proteins, which appear to function in concert to initiate and propagate tissue-specific biomineralization. Following enamel protein synthesis by ameloblast cells within the enamel organ, the subsequent steps of posttranslational modification, secretion, postsecretory processing and eventual removal of these proteins from forming enamel are largely unknown. To address this issue we have designed studies to investigate the hypothesis that enamel proteins are removed from enamel and translocated into the vasculature as relatively high-molecular-weight components. We examined enamel proteins recovered from serumless medium during prolonged organ culture of mouse capstage mandibular first molars. By 21 days in vitro the tooth crown formed and dentine and enamel biomineralization were apparent. At 31 days, explants retained metabolic activity and the enamel matrix showed extensive transformation. Immunologically identified enamel proteins of 26-18 k Da were produced by cultured tooth organs, translocated from tooth explants to the culture medium, recovered from the medium and then compared to control enamel protein from in vivo preparations. Comparable postsecretory processing of the 26-k Da amelogenin protein was observed in vitro and in vivo. We speculate that the pathway reported in the present studies is comparable to the processing of the enamel protein polypeptides of the maturing enamel which occurs in vivo. The in vitro organ culture model described in this report provides an approach with which to investigate the molecular events associated with epithelial-derived postsecretory processing of ECM molecules associated with tissue-specific biomineralization.     

The HIV-1 Vpu protein: a multifunctional enhancer of viral particle release

HIV accessory genes are expressed throughout the viral life cycle and regulate wide-ranging aspects of virus replication including viral infectivity (Vif and Nef), viral gene expression (Vpr) and progeny virion production (Vpu). While in many cases the molecular basis of accessory protein function is not fully understood, a consensus is emerging that these viral products are generally devoid of enzymatic activity and instead act as multifunctional adapters, subverting normal cellular processes to serve the needs of the virus. This review focuses on presenting our current knowledge of the HIV-1-specific Vpu protein and its essential role in regulating viral particle release, viral load and expression of the CD4 receptor.     

Altering biomineralization by protein design

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.     

Fluoride or/and aluminum induced toxicity in guinea pig teeth with the low expression of dentine phosphoprotein

This study investigated the damage and expression of dentine phosphoprotein (DPP) in guinea pig teeth by the administration of fluoride (F) or/and aluminum (Al). Fifty‐two guinea pigs were divided randomly into four groups (control, F, Al, and F+Al). F (150 mg NaF/L) or/and Al (300 mg AlCl₃/L) were added in their drinking water for 90 days. The levels of F ion, dentine sialophosphoprotein (DSPP) gene, and DPP protein in incisor and molar were determined, respectively. The results showed that the concentrations of F ion in F and F+Al groups were increased significantly. F induced the mottled enamel and irregular abrasion of teeth, which might occur as a consequence of depressed DSPP mRNA and DPP protein expression. Both the gene and protein expressions showed obvious decrease induced by Al, especially by F. There were no synergistic effects between F and Al, instead, Al inhibited the toxicity of F.     

利用古蛋白技术分析巨猿演化地位的评述

巨猿是中国南方更新世特有的大型猿类,因其巨大的牙齿和颌骨被认为是迄今生活在地球上体型最大的猿类。迄今为止,年代学和生物地层学证据显示巨猿的生存年代2~0.3 MaBP。由于中新世晚期—上新世时期化石记录的缺失,有关巨猿的起源和演化一直存在诸多争论。2019年,《自然》杂志报道广西吹风洞早更新世早期(1.9 MaBP)巨猿牙齿化石的古蛋白质研究^[1]。结果显示,巨猿牙齿牙釉质中保存了较为丰富的古代蛋白质,这些古蛋白质由409个特有的肽组成,分属6个内源性蛋白。对这些古老蛋白质的研究表明,巨猿在系统发育上属于猩猩分支系统,大约从10~12 MaBP前分化出来并独立演化。这是在亚热带地区的化石中首次提取如此古老的分子证据,提示古蛋白质研究有望为探索早期物种(包括人类)起源与演化提供重要依据。本文将简要回顾巨猿系统演化研究的历史,并对利用古蛋白技术分析巨猿的演化地位进行评述。     

Deciphering the cause of evolutionary variance within intrinsically disordered regions in human proteins

Why the intrinsically disordered regions evolve within human proteome has became an interesting question for a decade. Till date, it remains an unsolved yet an intriguing issue to investigate why some of the disordered regions evolve rapidly while the rest are highly conserved across mammalian species. Identifying the key biological factors, responsible for the variation in the conservation rate of different disordered regions within the human proteome, may revisit the above issue. We emphasized that among the other biological features (multifunctionality, gene essentiality, protein connectivity, number of unique domains, gene expression level and expression breadth) considered in our study, the number of unique protein domains acts as a strong determinant that negatively influences the conservation of disordered regions. In this context, we justified that proteins having a fewer types of domains preferably need to conserve their disordered regions to enhance their structural flexibility which in turn will facilitate their molecular interactions. In contrast, the selection pressure acting on the stretches of disordered regions is not so strong in the case of multi-domains proteins. Therefore, we reasoned that the presence of conserved disordered stretches may compensate the functions of multiple domains within a single domain protein. Interestingly, we noticed that the influence of the unique domain number and expression level acts differently on the evolution of disordered regions from that of well-structured ones.