Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelial nests.     

Regulation of amelogenin gene expression

The X-chromosomal amelogenin gene is expressed at a high level by ameloblast cells within the enamel organ for a short time during tooth development. Therefore, expression is both tooth specific and developmentally regulated. A Y-chromosomal amelogenin gene is also active in human and cow, but has not been detected in mouse. Genes and/or cDNAs have been cloned for mouse, human, cow, rat, pig, opossum, and hamster, and analyses have indicated that coding and upstream regions are conserved across species. Alternative splicing is extensive and produces as many as 9 mRNAs from the 7 exon murine gene, resulting from single and multiple exon skipping and alternate 3' site selection within exon 6. The pattern of alternative splicing varies both between species and during development, which is expected to result in some diversity through varying complements of amelogenin proteins associated with this highly conserved gene.     

Total variation norm for three-dimensional iterative reconstruction in limited view angle tomography

An iterative Bayesian reconstruction algorithm for limited view angle tomography, or ectomography, based on the three-dimensional total variation (TV) norm has been developed. The TV norm has been described in the literature as a method for reducing noise in two-dimensional images while preserving edges, without introducing ringing or edge artefacts. It has also been proposed as a 2D regularization function in Bayesian reconstruction, implemented in an expectation maximization algorithm (TV-EM). The TV-EM was developed for 2D single photon emission computed tomography imaging, and the algorithm is capable of smoothing noise while maintaining edges without introducing artefacts. The TV norm was extended from 2D to 3D and incorporated into an ordered subsets expectation maximization algorithm for limited view angle geometry. The algorithm, called TV3D-EM, was evaluated using a modelled point spread function and digital phantoms. Reconstructed images were compared with those reconstructed with the 2D filtered backprojection algorithm currently used in ectomography. Results show a substantial reduction in artefacts related to the limited view angle geometry, and noise levels were also improved. Perhaps most important, depth resolution was improved by at least 45%. In conclusion, the proposed algorithm has been shown to improve the perceived image quality.     

Evaluation of three-dimensional glenoid structure using MRI

The tilting angle and the shape of the glenoid cavity are considered to relate closely to shoulder stability. They are also important when planning arthroplasty and developing new designs. This study examines the glenoid cavity using 3-dimensional MRI. Forty volunteers (20 men, 20 women; average age 21.4; range 18–35 y) were enrolled in the study. The tilting angles of the glenoid bone were measured in 5 consecutive axial planes perpendicular to the glenoidal long axis. Cross sections were divided into 3 types (concave, flat, convex) according to the shape on each plane.
The average tilting angles for the 5 planes from the bottom to the top were 3.3±4.1, 1.4±3.8, −0.6±1.9, −1.4±3.3, and −6.2±3.3 degrees anteriorly, indicating that the 3-dimensional bony structure of the glenoid was twisted anteriorly to posteriorly. Images on the bottom plane consisted of 82.5% concave type, 15% flat type and 2.5% convex type, while only 3 cases (7.5%) showed concave at the top plane. The shape of the glenoid cavity is thought to be conducive to glenohumeral motion and stability.     

Secondary structure predictions for rat osteopontin

Five computerized methods were used to predict the secondary structure of osteopontin - a bone-derived cell attachment protein. The amino terminal one-fifth and the carboxy terminal one-third of the 301 amino acid protein contain eight alpha helices (41% of the total residues). The middle of the molecule contains a very acidic region of no predicted structure followed by two segments of beta structure which flank the cell attachment site of osteopontin. Examination of the amino acid sequence also revealed a potential calcium binding loop and two potential heparin binding sites.     

The effect of limited proteolysis by trypsin and chymotrypsin on bovine colostral IgG1.

Limited proteolysis of bovine colostral IgG1 by trypsin caused loss of specific antibody activity but column chromatography showed that relatively little cleavage into fragements had occurred. Polyacrlamide-agarose SDS electrophoresis of the 2-mercaptoethanol-treated digest revealed, however, that extensive cleavage of light chains had occurred even though most of the material before reduction had a mol. wt close to that of undigested IgG1. Although a Fab-type fragment was detected in the digest by immunoelectrophoresis it appeared to be only a minor component. Chymotrypsin had little effect upon either the structure or antibody activity of IgG1. These findings may explain the effect of trypsin and chymotrypsin on the bactericidal activity of colostral antibodies.     

The structure of infectious bovine rhinotracheitis virus

Summary The structure of infectious bovine rhinotracheitis virus was explored with the negative contrast technique. The virus was propagated in bovine testicle tissue culture cells. The essential components of the virus particles were: (1) the core, (2) the capsid, and (3) the envelope. The core measured 945 Å. The capsid consisted of 162 capsomeres and had an average diameter of 1085 Å. The capsomeres were arrayed in a highly regular pattern characteristic of 532 axial symmetry. The envelope varied in size but had an average diameter of 2000 Å.This work was supported in part by United States Public Health Service Research Grant AI -03820 from the National Institute of Allergy and Infectious Diseases.     

Evolutionary history of sex-linked mammalian amelogenin genes

Amelogenin (AMEL) arose prior to the emergence of tetrapods and transposed into an intron of the Rho GTPase-activating protein 6 gene. In the mammalian lineage leading to eutherians, a pair of homologous autosomes with this nested gene structure fused with the then already differentiating sex chromosomes by suppressing homologous recombination. As sex-chromosomal differentiation extended to the fused region, a pair of homologous AMEL genes too differentiated from each other in two steps; first in the 5' region (the promoter region to transposon MER5 in intron 2) and second in the remaining 3' region. This resulted in gametologous AMELX and AMELY in the eutherian sex chromosomes. Although the early differentiation of the 5' region between AMELX and AMELY is consistent with the lowered expression level of AMELY, there is no indication for deterioration of AMELY at the amino acid level. Rather, both AMELX and AMELY in particular lineages might undergo positive selection, followed by negative selection to preserve established function. Based on patterns and levels of AMELX and AMELY polymorphisms in the human population, it is also argued that a recombination cold spot near AMELX might be related to the cause of the ancient pseudoautosomal boundary.     

Secondary structure prediction of plant virus coat proteins

Application of the secondary structure prediction techniques to the coat protein amino acid sequences of turnip yellow mosaic virus and satellite tobacco necrosis virus yielded an excellent correlation with the patterns of secondary structural elements observed in the capsid proteins of southern bean mosaic virus and tomato bushy stunt virus. Helical predictions for the very basic N-terminal regions of three plant viruses suggested modes of protein-nucleic acid interaction similar to those proposed for histones and protamines.     

X-ray microtomographic imaging of three-dimensional structure of soft tissues

We report the x-ray microtomographic imaging of three-dimensional (3D) structure of soft tissues. The transparency of biological tissue to hard x-rays enables radiographic analysis of tissue entrails. However, biological tissues are mainly composed of light elements, which produce little contrast in a hard x-ray transmission image. Tissue structures were visualized by contrasting biological constituents with heavy elements. Efficient x-ray absorption by heavy-element dyes allowed the radiographic visualization of microstructures of soft tissues. The high-resolution computed tomography analysis provided the 3D microstructure of these microcontrasted tissues. Element-selective visualization of the stained tissue using x-ray absorption edges revealed the specific architecture of internal components. The structures obtained were used for rapid prototyping, giving 3D copies of human capillary vessels and fruit fly body.     

Helix structure of ribbon-like crystals in bovine enamel

In the early mineralized enamel crystals, ribbon-like crystals appear near the ameloblasts. Some ribbon-like crystals showed helical or spiral structure within restricted environment during the preparation of embryonic bovine specimens for electron microscope. These specimens did not suffer from the cutting damages nor staining effects. The main cause of the helix structure is considered a result of the dehydration during preparation. The periodic structure must reflect the regularity of initial enamel crystals. If dehydration caused the ribbon-like crystal to induce the periodic helix, it is one possibility that the earliest enamel crystal is OCP which has been proposed as a precursor of HA. Because it is considered that OCP is more sensitive to dehydration and more symmetric structure than biological HA. The periodicity of the helical ribbon-like structure was about 25 to 55 nm long and could be compared to the periodicity of organic helices which had observed in an immature rat enamel.     

The pH dependent amelogenin solubility and its biological significance

Amelogenins are a group of extracellular enamel matrix proteins which are believed to be involved in the regulation of the size and habit of enamel crystals. The aim of this study was to compare the solubility properties of several amelogenins in various pH (4.0-9.0) solutions with an ionic strength (IS) of 0.15 M using the Micro BCA protein assay at 25 degrees C or 37 degrees C. The solubility of the recombinant amelogenin rM179 was lowest (0.7 mg/ml) close to its isoelectric point and it increased below and above this point. The solubility of the recombinant amelogenin rM166 remained almost the same (1-2 mg/ml) as the pH rose from 6.0 to 9.0 and it increased as the solution became more acidic. Synthetic "tyrosine-rich amelogenin polypeptide" (TRAP) was extremely insoluble (<0.2 mg/ml) in the pH range studied while synthetic "leucine-rich amelogenin polypeptide" (LRAP) was readily soluble (>3.3 mg/ml). The native porcine amelogenin with apparent molecular weight 25 kDa shared similar solubility behavior to rM179. The porcine 23 kDa amelogenin was only sparingly soluble (0.3-0.8 mg/ml) over a wide range of pH. Interestingly, the porcine 20 kDa amelogenin was remarkably soluble in the pH range of 4.0 to 6.0 (approximately 12 mg/ml), but the solubility dropped strikingly to only approximately 0.2 mg/ml at pH larger than approximately 7.0. The strong dependence of amelogenin solubility on solution pH may be involved in the regulation of aggregation, enzymatic degradation and the binding properties of amelogenins, thus playing an important role in enamel biomineralization.     

A digital three-dimensional atlas of structure/function relationships

The techniques and issues relevant to the development of a digital three-dimensional atlas of brain structure and function are discussed. The goals were to develop the appropriate methods for an interactive and quantitative stereotactic atlas to describe both anatomy and physiology. The work presented here used serial sections from unstained, thionein-stained and 2-deoxyglucose autoradiographic data sets.     

Of mice and men: anatomy of the amelogenin gene

Mammalian enamel matrix is composed of two principal proteins, the enamelins and amelogenin. Recombinant complementary DNA (cDNA) molecules for the predominant mouse amelogenin have been identified, characterized by direct determination of the DNA sequence, and used as a specific hybridization probe. The spatial- and temporal-restricted pattern for amelogenin gene expression within developing mouse molars has been traced at the level of a single cell using in situ hybridization. The mouse genome has been shown to contain only one copy of the amelogenin (AMEL) gene which is not amplified or rearranged during ameloblast determination. In contrast, the human genome contains two copies of the AMEL gene, one residing on the X chromosome and one upon the Y chromosome. These observations, the availability of specific enamel gene probes coupled with the application of new techniques in molecular biology now afford unique opportunities for the analysis of the molecular basis of inherited defects of human enamel such as amelogenesis imperfecta. Recent advances towards obtaining a physical map and the complete nucleotide sequence for the human genome, as well as the documented developmental biology, defined genetics and transgenic capability of the mouse, suggest that mouse and man are the most relevant and potentially informative models for analysis of normal and abnormal enamel biomineralization.     

Secondary structure of poliovirus RNA: correlation of computer-predicted with electron microscopically observed structure

A secondary structure map of poliovirus 1, strain Mahoney, RNA was determined by psoralen crosslinking the (+) strand and visualizing the structures in the electron microscope. Hairpins and looped hairpins were observed, and the size and distribution were measured. To orient map features the 3' end of the RNA was linked to polybromodeoxyuridine [poly(BUdR)]SV40 and histograms were constructed from these measurements. Secondary structure maps of the RNA were likewise constructed from the results of computer prediction programs for secondary structure. The programs used were those of M. Zuker (RNA2 and FOLD) which calculate a minimal global energy for a given sequence. Many single hairpins predicted by both RNA2 and FOLD showed a correlation with the histograms of hairpin structures of RNA crosslinked with psoralen. A secondary structure map was also constructed for the entire 7433 bases using the option in FOLD which allows multi-branch loops by folding uniformly stepped overlapping segments. Any structure that occurred at or greater than a given frequency was selected and mapped with respect to genome position. A correlation in structured regions was seen between psoralen-derived and computer-predicted maps of secondary structure. Furthermore, a region of large loops from base position 681 to 3899 was noted that corresponded to frequently observed large loop(s) in electron micrographs of psoralen preparations of RNA. Agreement between the two methods of determining secondary structure strengthens the credibility of the computer-aided methods used for predicting secondary structure and allows us to suggest an overall secondary structure map for poliovirus RNA.