Derived protein and cDNA sequences of hamster amelogenin

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3′ rapid amplification of cDNA ends (3′ RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3′ non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5′ PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.     

Expression and characterization of a Rana pipiens amelogenin protein

Amelogenin, the major protein of developing enamel matrix, controls enamel crystal growth via unique supermolecular features. While much has been contributed to our understanding of mammalian amelogenin function, little is known about how amelogenin and its unique physico-chemical features have evolved among vertebrates. Here we report, for the first time, amphibian amelogenin recombinant protein expression and characterization in Rana pipiens . In order to characterize R. pipiens amelogenin, the newly discovered amelogenin coding sequence was amplified, subcloned, and expressed in Eshcerichia coli . Our newly generated R. pipiens amelogenin-specific antisera resolved a major 19-kDa band on western blots of frog tooth extracts and revealed an enamel organ tissue-specific localization pattern using immunohistochemistry. Using mass spectroscopy, a single major compound with a molecular weight of 21.6 kDa was detected, which corresponded to the amino acid sequence-based molecular weight prediction of the His fusion recombinant protein. Dynamic light scattering studies resolved 41-nm radius subunits compared with 14-nm radius subunits from mouse recombinant amelogenin controls. Transmission electron microscopy revealed defined spherical subunits in R. pipiens matrix self-assembly in contrast with a homogeneous 'stippled' matrix in mouse amelogenin matrix self-assembly. Our data suggest that R. pipiens amelogenin is distinguished from mammalian amelogenins by a number of unique physico-chemical properties which may be related to specific modes of crystal formation in frog enamel.     

口腔鳞状细胞癌组织中PTEN基因第5、8外显子突变的检测

目的:探讨口腔鳞状细胞癌组织中PTEN基因第5、8外显子突变的情况,及其与口腔鳞癌发生发展的关系.方法:对32例鳞癌组织应用聚合酶链反应-单链构象多态性分析,检测PTEN基因第5、8外显子的突变情况.结果:PTEN基因第5、8外显子全长序列PCR扩增均获成功;第5、8外显子均未发现有突变.结论:提示口腔鳞癌的发生可能和PTEN基因第5、8外显子的突变无关.     

Expression of odontogenic ameloblast-associated protein, amelotin, ameloblastin, and amelogenin in odontogenic tumors: immunohistochemical analysis and pathogenetic considerations

Screening for expression of amelogenesis-related proteins represents a powerful molecular approach to characterize odontogenic tumors and investigate their pathogenesis. In this study, we have examined the presence and distribution of odontogenic ameloblast-associated protein (ODAM), amelotin (AMTN), ameloblastin (AMBN), and amelogenin (AMEL) by immunohistochemistry in samples of adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), developing odontoma, ameloblastoma, calcifying cystic odontogenic tumor (CCOT), ameloblastic fibroma (AF), myxoma, odontogenic fibroma (OF), and reduced enamel epithelia (REE). Positive results were obtained in those tumors with epithelial component, except for AF, OF, and ameloblastoma. ODAM was found around mineralized structures (dystrophic calcifications) and CEOT's amyloid, whereas AMTN stained the eosinophilic material of AOTs. The CCOT transitory cells to ghost cells were strongly positive with all proteins except AMEL, and the REE as well as odontomas showed immunoexpression for ODAM, AMTN, AMBN, and AMEL similar to those found in normal rat tooth germs. Based on these results, some histopathogenetic theories were formulated.     

The amelogenin story: origin and evolution

Genome sequencing and gene mapping have permitted the identification of HEVIN (SPARC-Like1) as the probable ancestor of the enamel matrix proteins (EMPs), amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). We have undertaken a phylogenetic analysis to elucidate their relationships. AMEL genes available in databases, and new sequences obtained in blast searching genomes or expressed sequence tags, were compiled (22 full-length sequences), aligned, and the ancestral sequence calculated and used to search for similarities using psi - blast . Hits were obtained with the N-terminal region of AMBN, ENAM, and HEVIN. We retrieved all available AMBN ( n  = 8), ENAM ( n  = 3), and HEVIN ( n  = 4) sequences. The sequences of the four proteins were aligned and analyzed phylogenetically. AMEL and AMBN are sister genes, which diverged after duplication of a common ancestor issued from ENAM . The latter derived from a copy of HEVIN . Comparisons of gene organization, amino acid sequences and location of ENAM and AMBN , adjacent on the same chromosome, suggest that AMBN is closer to ENAM than AMEL . This supports AMEL as being derived from AMBN duplication. This duplication occurred long before tetrapod differentiation, probably in an ancestral osteichthyan. The story of AMEL origin is completed as follows: SPARC → HEVIN → ENAM → AMBN → AMEL .     

Tissue distribution and nucleotide sequence of bovine mRNA for salivary proline-rich protein P-B

The tissue distribution of P-B was investigated to obtain information on the physiological significance of this proline-rich protein. To design primers and probes for a tissue distribution analysis, a polymerase chain reaction (PCR)-based cloning of bovine P-B cDNA was performed using tooth germ and the nucleotide sequence was determined. The cloned bovine P-B cDNA was composed of 356 bp and included the region corresponding to the mature P-B protein and part of the 3' non-coding sequence. This part of the sequence is identical to the corresponding region of human P-B cDNA from the submaxillary gland. DNA corresponding to the P-B mRNA was amplified by PCR using cDNAs from various bovine tissues including tooth germ, submaxillary gland, parotid gland, lachrymal gland, heart, liver, stomach, pancreas, spleen, kidney, adrenal, and ovary. A quantitative analysis indicated the heart, submaxillary gland, tooth germ and kidney to be major sites of P-B expression. The ubiquitous distribution of P-B mRNA among bovine tissues together with findings of the presence of genes hybridizable with a DNA probe for P-B among species such as human, bovine, rat, mouse, and yeast as reported previously suggested a fundamental physiological role for this protein.     

釉原蛋白基因启动子的克隆及在不同细胞中转录调控的分析

目的：克隆釉原蛋白基因启动子及可能影响转录调控的序列,分析其在不同细胞中的转录模式。方法：检索并分析釉原蛋白基因的上游调控序列．利用PCR及酶切方法,从小鼠C57BL／6J基因组上扩增不同长度（包括基本启动子区域）的转录调控序列,与PGL3-Basic载体的虫荧光素酶基因连接。瞬时转染CHO、Hela、UMR-106细胞,检测荧光素酶的活性．分析不同长度的启动子片段在各种细胞中的转录活性。结果：共构建了6个不同长度的报告载体,瞬转后发现在Hela细胞中有较强的荧光素酶活性,在CHO、UMR-106细胞中活性很弱。在不同的细胞内,启动子活性随片段长度变化,其变化趋势有明显的相似性。表现为在转录起始点上游975bp与532bp的区域具有较强的转录活性．而转录起始点上游285bp区域的转录活性有所降低。结论：釉原蛋白的启动子可在Hela细胞中激活,Hela细胞可作为研究釉原蛋白启动子转录调控的细胞模型：初步判断釉原蛋白启动子上的-1693与-975之间的序列为转录抑制区域,-532与-285之间为转录增强区域。     

小鼠Q9D3J8(釉成熟蛋白)基因在磨牙牙胚中的表达

目的:报道一新发现的成熟期成釉细胞特异性表达蛋白:釉成熟蛋白(MASP,m ature am elob last-spec ificprote in)。方法:利用生物信息学工具和基因数据库分析位于人4号染色体4q13.3区域的新基因UNQ689;小鼠Q9D3 J8基因为UNQ689的同源基因。根据小鼠Q9D3 J8的基因序列设计引物,扩增长度为982bp的基因片段:分离小鼠下颌第一磨牙牙胚及周围组织,采用RT-PCR技术观察小鼠Q9D3 J8基因在小鼠下颌磨牙组织中的表达。制备D igoxygen in标记的小鼠Q9D3 J8 cRNA探针,以小鼠下颌第一磨牙及其周围组织为研究标本,采用原位杂交技术对Q9D3 J8基因表达进行组织学定位。结果:利用生物信息学工具和基因数据库,发现am elob lastin基因上游一未报道的新基因UNQ689/Q9D3 J8。在染色体基因图谱位置关系上,UNQ689/Q9D3 J8,am elob lastin和enam elin基因紧密相连。RT-PCR产物的琼脂糖电泳显示Q9D3 J8基因在出生后10d和15d的小鼠磨牙组织中表达明显。原位杂交研究显示Q9D3 J8基因仅在小鼠磨牙成熟期成釉细胞中呈特异性表达。结论:本研究提示UNQ689/Q9D3 J8基因为成熟期成釉细胞特异性表达蛋白基因;在染色体DNA上,UNQ689/Q9D3 J8,am elob lastin和enam elin共同组成一个成釉细胞特异性表达蛋白基因串。根据UNQ689/Q9D3 J8基因在组织中的分布和表达,我们暂时将UNQ689/Q9D3 J8基因命名为MASP(釉成熟蛋白)基因。     

牛牙骨质附着蛋白N末端氨基酸序列分析

目的 :对牛牙骨质附着蛋白进行N末端氨基酸序列分析 ,为牙骨质附着蛋白的克隆表达提供一定的理论依据。方法 :应用乙酸和盐酸胍提取法获取牛牙骨质附着蛋白 ,鉴定纯度后进行N末端氨基酸序列分析。结果 :所获得的牛牙骨质附着蛋白纯度 >95 % ,N末端 15个氨基酸残基为 :Ile、Pro、Leu、Asp、Pro、Val、Ala、Gly、Cys、Lys、Glu、Pro、Ala、Ala、Asp。 结论 :测得的氨基酸序列具有特异性。     

Putative heat shock protein 70 gene from Actinobacillus actinomycetemcomitans: molecular cloning and sequence analysis of its gene

We have cloned and sequenced two overlapping fragments of chromosomal DNA from Actinobacillus actinomycetemcomitans. The nucleotide sequence contained two open reading frames. The deduced amino acid sequences of the two open reading frames showed significant homology with the heat shock proteins hsp70 and hsp40 of other organisms respectively. The upstream open reading frame consisted of 1902 bp, corresponding to 634-amino-acid residues. The CAA codon for glutamines was frequently seen in hsp70, i.e., in 30 of 32 glutamines (93.8%). The spacing region between the two open reading frames was unusually long compared with other prokaryotic organisms. A number of unique and distinguishing features of the sequences in the hsp70 family were verified, and it was found that a particular spacing sequence between the hsp70 and hsp40 gene loci can be used to identify A. actinomycetemcomitans from the periodontal pocket.     

Mutation analysis of the MSX1 gene exons and intron in patients with nonsyndromic cleft lip and palate

Cleft lip with or without cleft palate and cleft palate (CL/CLP/CP) is one of the most common malformations among newborns. The estimated prevalence in Latvia is 1/700. Nonsyndromic CL/CLP/CP is a complex trait determined by multiple, interacting genetic and environmental factors. MSX1 gene is one of the most important candidate-genes, which had been analyzed in relation with nonsyndromic CL/CLP/CP. The objective of our study was to examine the etiologic role of MSX1 gene mutations in the development of nonsyndromic CL/CLP/CP in Latvian population. MATERIALS AND METHODS: DNA was extracted from venous blood of 53 patients with cleft lip with or without palate. Polymerase chain reaction (PCR) was performed of selected segments of MSX1 gene. These were sequenced and analysed by comparison with reference sequence, accession Nr. AF426432 (NCBI). RESULTS: 16 DNA sequence variations were identified in 53 patient samples; 6 of them have not been previously described. Identified sequence variations localized in coding regions do not cause amino acid substitutions, therefore they are not considered as mutations with an etiological role in CL/CLP/CP development. Baltic-Taiwan joint research project "Identification of genes involved in craniofacial morphogenesis and susceptibility to orofacial clefting in a human genome scan 2004-2006".     

Levels of gingival crevicular metalloproteinases-8 and -9 in periodontitis

Matrix metalloproteinases (MMPs), the key enzymes responsible for matrix degradation, are derived from polymorphonuclear leukocytes during the early stages of periodontitis. The aim of this study was planned to determine the levels of GCF (gingival crevicular fluid) matrix metalloproteinase-8 (MMP-8) and metalloproteinase-9 (MMP-9) patients with periodontitis and in healthy controls. Levels of crevicular MMP-8 and -9 were determined by ELISA in subjects with healthy without any periodontal disease (n = 10) and periodontitis (n = 10). Significantly higher crevicular MMP-8 and -9 were observed in cases of periodontitis compared to healthy adults. Crevicular MMP-8 and -9 may serve as biomarkers of periodontal disease and aid in early detection of periodontitis.     

CD14基因重组和序列分析

目的：对膜CD14基因进行截短突变和真核表达优化重组，以期得到可溶性CD14全长和最小功能段基因。方法：根据设计合成引物，以膜CD14基因为模板，PCR扩增目的基因，亚克隆入pGEM-T载体，进行序列分析。结果：PCR扩增分别得到1.1和0.5kb的2个基因片段，大小和序列同预期一致。结论：通过PCR扩增获得重组可溶性CD14全长及最短功能段基因，为其结构和功能研究奠定了基础。     

The genetic relatedness of Porphyromonas gingivalis clinical and laboratory strains assessed by analysis of insertion sequence (IS) element distribution

OBJECTIVES: Porphyromonas gingivalis is frequently found in periodontitis lesions. This organism contains a large number of insertion sequence (IS) elements. We sought to determine the distribution of seven IS elements from strain W83 among nine P. gingivalis laboratory strains and nine clinical isolates and to use these findings to determine strain relationships. METHODS: Southern blots of BamHI digested genomic DNA digests were probed with insertion sequence elements ISPg1-7. RESULTS: The restriction fragment length polymorphism (RFLP) patterns revealed that five of the nine laboratory strains, including strain W83, were nearly identical for all seven IS elements. Two of nine clinical isolates were similar to the five laboratory strains. Two of the four remaining laboratory strains had similar or identical RFLP patterns. The remaining two laboratory strains had limited similarity to clinical strains. Four of the clinical isolates had identical RFLP patterns for all seven IS elements. The three remaining clinical isolates were unique in their RFLP patterns. Several strains lacked from one to four of the IS elements. Similar strain relationships were suggested regardless of the IS element examined. CONCLUSIONS: Transposition and recombination between IS elements are not sufficiently pervasive to obscure strain relationships, though this does not preclude the possibility that such events play an important role in allowing P. gingivalis to adapt to new environments. Given the level of genetic diversity observed, it may be especially important to examine genetically diverse strains when drawing conclusions based on the W83 P. gingivalis genomic database.     

Overdenture retaining bar stress distribution: A finite-element analysis

Abstract

Objective. Evaluate the stress distribution on the peri-implant bone tissue and prosthetic components of bar-clip retaining systems for overdentures presenting different implant inclinations, vertical misfit and framework material. Materials and methods. Three-dimensional models of a jaw and an overdenture retained by two implants and a bar-clip attachment were modeled using specific software (SolidWorks 2010). The studied variables were: latero-lateral inclination of one implant (–10°, –5°, 0°, +5°, +10°); vertical misfit on the other implant (50, 100, 200 µm); and framework material (Au type IV, Ag-Pd, Ti cp, Co-Cr). Solid models were imported into mechanical simulation software (ANSYS Workbench 11). All nodes on the bone’s external surface were constrained and a displacement was applied to simulate the settling of the framework on the ill-fitted component. Von Mises stress for the prosthetic components and maximum principal stress to the bone tissue were evaluated. Results. The +10° inclination presented the worst biomechanical behavior, promoting the highest stress values on the bar framework and peri-implant bone tissue. The –5° group presented the lowest stress values on the prosthetic components and the lowest stress value on peri-implant bone tissue was observed in –10°. Increased vertical misfit caused an increase on the stress values in all evaluated structures. Stiffer framework materials caused a considerable stress increase in the framework itself, prosthetic screw of the fitted component and peri-implant bone tissue. Conclusions. Inclination of one implant associated with vertical misfit caused a relevant effect on the stress distribution in bar-clip retained overdentures. Different framework materials promoted increased levels of stress in all the evaluated structures.