Qualitative changes in protein synthesis associated with polyspermic fertilization of human eggs

To investigate the early molecular events in human oocytes that are triggered by fertilization, the authors examined the pattern of polypeptides synthesized by unfertilized and dispermic embryos obtained through an in vitro fertilization and embryo transfer (IVF-ET) program. Compared with unfertilized oocytes of the same postovulatory age, the de novo protein synthesis in tripronuclear dispermic zygotes (21 hours postinsemination) was characterized by the appearance of three novel protein bands with molecular weights of 41.2, 35.3, and 26.0 kD. Concomitant with these changes, these zygotes showed the disappearance of bands at 54.0, 36.5, and 28.0 kD, along with the decreased synthesis of a protein band at 42.5 kD. Although 24% of the aged unfertilized oocytes exhibited bands corresponding to 41.2 and 35.3 kD, the 26.0 kD protein is restricted to the tripronuclear embryos. The significance of these results is discussed in relation to the use of polyspermic human oocytes as a model for the study of the early molecular events triggered by fertilization.     

Ultrastructure of the human enamel organ

Summary The fine structure of external enamel epithelium, stellate reticulum and stratum intermedium in primary tooth germs (bell stage) from four human foetuses was investigated.Characteristically, the cells of the differentiated external enamel epithelium, stellate reticulum and stratum intermedium exhibit many free ribosomes, few rough endoplasmic reticulum cisterns, well-developed Golgi complexes, many coated and smooth vesicles, often in relation to the cell membranes, and many bundles of tonofilaments. The cells are connected by numerous desmosomes and gap junctions.A parallel differentiation of stratum intermedium — external enamel epithelium, and the ameloblast layer is demonstrated.The morphology of the cells of the three layers indicates that these have secretory, transport and supporting functions.     

Identification of protein components in in vivo human acquired enamel pellicle using LC-ESI-MS/MS

The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integument relies on efficient pellicle harvesting and protein identification procedures. Material from three individual subjects and two pooled samples yielded the identification by LC-ESI-MS/MS of 130 pellicle proteins of which 89 were found in three or more experiments. A high intersubject consistency in pellicle composition was observed.     

Sheath configurations in human cuspal enamel

To determine the prism sheath configurations in human cuspal enamel 80 teeth were initially ground to produce flat surfaces through the following planes: a horizontal series at successively greater distances from the dentinoenamel junction and longitudinally through the center of the cusps. Individual teeth were suspended in an acid-alcohol solution (1 cm³ conc. HCl in 100 cm³ 95% ethanol) at 37°C for seven to ten days. The treatment “softened” the enamel to a depth of approximately 1 mm. The teeth were embedded in Epon and sectioned at 0.5 to 10 μm with a diamond knife. Thick and thin ground sections for phase contrast microscopy and acid-etched ground sections for Nomarski differential interference microscopy were prepared through the same regions. In thicker longitudinal sections, the prisms in gnarled enamel formed a zig-zag pattern which was unlike the twisting pattern generally observed in ground sections. The thinnest transverse sections showed the sheath outlines to be dramatically different from those seen elsewhere in the enamel. Some prism sheaths were circular, others were in the form of spirals. What could be described as sheaths within sheaths were also seen. In the thinnest longitudinal sections the prisms were seen to be elongated and discontinuous. Sheath outlines in enamel adjacent to the central core of gnarled enamel were similar to those described elsewhere in the body of the enamel. Keyhole, modified keyhole patterns and arcade forms were the dominant sheath patterns. Other atypical sheath configurations were seen scattered throughout this region.     

Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches

Precursor proteins of the acquired enamel pellicle derive from glandular and non-glandular secretions, which are components of whole saliva. The purpose of this investigation was to gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize separation and resolution using only micro-amounts of protein, a two-dimensional gel electrophoresis system was employed. Protein samples from parotid secretion, submandibular/sublingual secretion, whole saliva, and pellicle were subjected to isoelectric focusing followed by SDS-PAGE. Selected protein spots were excised, subjected to "in-gel" trypsin digestion, and examined by mass spectrometry (MS). The data generated, including peptide maps and tandem MS spectra, were analyzed using protein data base searches. Components identified in whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle included histatins, lysozyme, statherin, cytokeratins, and calgranulin B. The results showed that whole saliva and pellicle have more complex protein patterns than those of glandular secretions. There are some similarities and also distinct differences between the patterns of proteins present in whole saliva and pellicle. MS approaches allowed identification of not only well characterized salivary proteins but also novel proteins not previously identified in pellicle.     

Variation in modern human enamel formation times

Most of what we know about the timing of human enamel formation comes from radiographic studies on children of known age. Here, we present new longitudinal data derived from a histological analysis of tooth enamel. Two samples, one from southern Africa and one from northern Europe, contained all anterior and molar tooth types. Two further samples contained only one tooth type: canines from a medieval Danish sample and third molars from a modern North American sample. Data were collected on 326 molars and 352 anterior teeth. Each tooth was sectioned and prepared for polarized light microscopy. We used daily enamel cross striations to determine cuspal enamel formation time, recorded the periodicity of long-period striae in the lateral enamel, and used this value to calculate enamel formation times for each decile of crown length. We present data that reveal some of the processes whereby differences in enamel formation times arise between our samples. Mean cuspal enamel formation times were similar in southern African and northern European anterior teeth, but differed in certain molar cusps. All the southern African anterior teeth completed enamel formation earlier. The greatest difference in mean chronological age at enamel completion was 5.2 vs. 6.2 years of age in lower canines. However, enamel completion times in the molar teeth showed few differences between the samples, with mean times for the longest forming cusps all falling between 3.0 years and 3.45 years. Our data suggest fewer differences between samples and smaller ranges of variation than in many radiographic studies and present a more realistic picture of worldwide variation in enamel formation times.     

Validation of mechanically-assisted sodium dodecyl-sulphate elution as a technique to remove pellicle protein components from human enamel

The salivary film, denoted the pellicle, formed on oral surfaces is of great importance for oral health and comfort. The present study describes mechanically-assisted sodium dodecyl sulphate (SDS) elution of the in vivo pellicle formed on human enamel and visualisation of the desorbed pellicle proteins using two-dimensional gel electrophoresis (2-DE). To verify this removal of the pellicle, a combined mechanical and surfactant procedure was additionally performed on an in vitro pellicle formed on human enamel, and the effectiveness was validated by mechanical removal in combination with HCl. As indicated by protein quantitation and one dimensional gel electrophoresis, rubbing with polyamide fibre pellets soaked in a 0.5% SDS solution was optimal for completely removing the adsorbed proteins from the enamel surface, and yet provided separation of the proteins by 2-DE to enable identification in future studies.     

Validation of mechanically-assisted sodium dodecyl-sulphate elution as a technique to remove pellicle protein components from human enamel

The salivary film, denoted the pellicle, formed on oral surfaces is of great importance for oral health and comfort. The present study describes mechanically-assisted sodium dodecyl sulphate (SDS) elution of the in vivo pellicle formed on human enamel and visualisation of the desorbed pellicle proteins using two-dimensional gel electrophoresis (2-DE). To verify this removal of the pellicle, a combined mechanical and surfactant procedure was additionally performed on an in vitro pellicle formed on human enamel, and the effectiveness was validated by mechanical removal in combination with HCl. As indicated by protein quantitation and one dimensional gel electrophoresis, rubbing with polyamide fibre pellets soaked in a 0.5% SDS solution was optimal for completely removing the adsorbed proteins from the enamel surface, and yet provided separation of the proteins by 2-DE to enable identification in future studies.     

Some observations on the trace element concentrations in human dental enamel

The concentration of trace elements has been measured for dental enamel from 86 healthy human teeth using particle-induced X-ray emission (PIXE). The majority of the teeth (n = 70) were collected from dentists in the county of Oxfordshire in the United Kingdom, although a smaller group (n = 16) were collected from Cornwall. The elements K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Sr, Pb, and Hg have been detected and statistically analyzed by grouping according to sex, age, and geographical location. The concentrations of Fe and Cu were found to be lower in the teeth from female donors (P < 5%) and are believed to result from the continued burden of blood loss during menstruation. Strong positive correlations (P < 0.1%) were found between Ca, Co, Ni, and Zn for all groups; these elements were also found to exhibit a negative correlation (P < 1%) with age for teeth from female donors. This is believed to be related to decalcification during the menopause. Pb was found to exhibit a positive correlation (P < 5%) with age for both sexes, and is believed to substitute for Ca in the Ca hydroxy apatite (HAP) within the dental enamel.