Determination of Age from Cemental Incremental Lines for Forensic Dentistry

Determination of age from cemental incremental lines was evaluated in intact teeth obtained from 17 individuals aged 23-77 years. Mineralized 100 μm cross sections were subjected to one of three treatments: unstained, stained with Villanueva's blood stain, and stained with acridine orange. Ideal areas were selected by light microscopy and photographed. Countability of incremental lines from photographic enlargements were evaluated. The average number of years required for the eruption of a particular tooth was added to the incremental lines count to determine the estimated age for that individual. Results obtained from unstained mineralized 100 μm thick cross sections using differential interference microscopy (Nomarsky) provided the most countable lines. The accuracy and repeatability of the method is not dependent on tooth type or location, but on the average obtained from making as many counts as possible. This method can be applied to general populations regardless of systemic or periodontal health.     

A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

Evaluation of Electron Microscopic Sample Preparation Methods and Imaging Techniques for Characterization of Cell-Mineral Aggregates

Scanning Electron Microscopy (SEM) is used to image geomicrobiological samples, typically containing interfaces between “hard and soft materials” such as minerals and cells, which represent challenges for artifact-free preparation for high-resolution imaging. We used cell-mineral aggregates produced during microbial Fe(II) oxidation and Fe(III) reduction to evaluate different sample preparation and imaging techniques. Both rapid freezing and standard critical point drying (CPD) preserve structures of geomicrobiological samples, at least the ones obtained for Fe(II)-oxidizing and Fe(III)-reducing bacteria, without artifacts. We recommend a SEM sample preparation scheme for geomicrobiological specimens and discuss critical parameters like fixation, dehydration, coating, and acceleration voltages.     

Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Enzyme, Lectin and Immunohistochemistry of Plastic Embedded Undecalcified Bone and other Hard Tissues for Light Microscopic Investigations

Enzymes and tissue antigens were localized on plastic embedded undecalcified bones and teeth using Technovit 7200 VLC (Kulzer, Germany). This resin is hard enough for cutting and grinding procedures on rotating plates with diamond layers. The pores between the diamond grains are not obstructed with this resin. The procedure described here permits localization of antigens in the soft tissues adjacent to, or in the biological hard tissues themselves and in dental implants (ceramic or metallic) on the light microscopic level. The undecalcified bone is fixed and embedded in plastic and cut at 100-150 μm. The slices are ground automatically by a grinding machine to a thickness of 5-10 μm. After application of the substrates for alkaline and acid phosphatases and the required dyes, the distribution of these enzymes can be demonstrated. Tissue antigens also can be detected with slightly modified standard techniques of immunohistochemistry and lectin histochemistry using the peroxidase technique or fluorescence microscopy.     

Biologic,Immunocytochemical, and Cytogenetic Characterization of Two New Human Melanoma Cell Lines: IIB-MEL-LES and IIB-MEL-IAN

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD₃ and GD₂ gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10,20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.     

Content and Distribution of Noncollagenous Matrix Proteins in Bone and Cementum: Relationship to Speed of Formation and Collagen Packing Density

The organic matrix of collagen-based calcified tissues consists of a supporting collagen meshwork and various noncollagenous matrix proteins (NCPs). Together, they contribute to determining the structure and biomechanical properties of the tissue. Their respective organization and interrelation can advantageously be examined by immunocytochemistry, an approach which allows correlation of composition with structure. The aim of this article is to review postembedding immuno- and lectin-gold-labeling data on the characterization of the noncollagenous compartment in rat and human bone and cementum, and on its relationship to collagen. The two major NCPs, bone sialoprotein and osteopontin, generally codistribute and accumulate in cement lines and in the spaces among the mineralized collagen fibrils. However, there are variations in their distribution and density of labeling throughout the tissue. Indeed, bone and cementum can form in environments that are either poor or enriched in NCPs. The amount of NCPs generally correlates with bone and cementum types and with speed of formation of the tissue and packing density of collagen fibrils. Taken together, the data suggest that production of both collagenous and noncollagenous constituents can be "modulated" during formation of collagen-based calcified tissues. It is concluded that, in addition to structural and compositional parameters, tissue dynamics must be taken into consideration in order to understand the significance of the apparent accumulation of NCPs at some sites and to determine the mechanisms of normal and pathological calcified tissue formation.     

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca²⁺-sensitive dyes is used for measurement of spatial and temporal aspects of Ca²⁺ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca²⁺-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca²⁺-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca²⁺ response as % change in fluorescence is obtained.     

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts ^{1 2}. Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) ^{3 4}. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.     

Developmental Aspects of Sexual Dimorphism in Hominoid Canines

We examined the histology of canine teeth in extant hominoids and provided a comparative database on several aspects of canine development. The resultant data augment the known pattern of differences in aspects of tooth crown formation among great apes and more importantly, enable us to determine the underlying developmental mechanisms responsible for canine dimorphism in them. We sectioned and analyzed a large sample (n = 108) of reliably-sexed great ape mandibular canines according to standard histological techniques. Using information from long- and short-period incremental markings in teeth, we recorded measurements of daily secretion rates, periodicity and linear enamel thickness for specimens of Pan troglodytes, Gorilla gorilla, Pongo pygmaeus and Homo sapiens. Modal values of periodicities in males and females, respectively, are: Pan 7/7; Gorilla 9/10; Pongo 10/10; and Homo 8/8. Secretion rates increase from the inner to the outer region of the enamel cap and decrease from the cuspal towards the cervical margin of the canine crown in all great ape species. Female hominoids tend to possess significantly thicker enamel than their male counterparts, which is almost certainly related to the presence of faster daily secretion rates near the enamel-dentine junction, especially in Gorilla and Pongo. Taken together, these results indicate that sexual differences in canine development are most apparent in the earlier stages of canine crown formation, while interspecific differences are most apparent in the outer crown region. When combined with results on the rate and duration of canine crown formation, the results provide essential background work for larger projects aimed at understanding the developmental basis of canine dimorphism in extant and extinct large-bodied hominoids and eventually in early hominins.     

用免疫荧光标记对人组织中肥大细胞亚型的分选与形态观察

方法:利用中性蛋白酶成分、特征性酶抗体的免疫荧光染色和流式细胞仪确定分选肥大细胞亚型,以激光扫描共聚焦显微镜显示肥大细胞内分泌颗粒。结果:三种免疫表型被确定:肥大细胞的类胰蛋白酶阳性(MCT)、类糜蛋白酶阴性;类糜蛋白酶阳性(MCC)、类胰蛋白酶阴性和类胰蛋白酶阳性、类糜蛋白酶阳性(MCTC)。肥大细胞内分泌颗粒分散或聚集存在,分泌颗粒突起分泌或以分散的方式释放。分泌颗粒大范围释放后,肥大细胞的形态结构发生了改变。结论:利用肥大细胞的特征性酶抗体、免疫荧光标记和流式细胞仪可将人组织中的肥大细胞分选纯化为三种亚型;以共聚焦显微镜显示肥大细胞含有丰富的分泌颗粒,它说明肥大细胞具备了为人体I型变态反应提供快速反应的物质基础。     

Light and Electron Microscopic Observations on Encystment of Acanthamoeba palestinensis, Reich

SYNOPSIS. The ultrastructural changes occurring during encystment of Acanthamoeba palestinensis have been investigated. The cyst wall consists of endocyst and exocyst, both having the same fine structure. At irregular intervals in the cyst wall ostioles occupied by opercula are present. The nuclear membrane forms bulb-shaped projections and releases vesicles bounded by double membranes into the cytoplasm. Dense nucleolus-like bodies of different sizes and variable numbers are found in the nucleus of every cyst. The importance of the cyst structure as a taxonomic criterion is discussed.     

Effects of Inorganic Oxides,Sulfides, Chlorides,and Acid Chlorides on Metachromasia and Other Staining Properties

The ability of 17 inorganic compounds (POCl₃, PSC1₃, PC1₃, P₂O₅, P₂S₅, P₄S₃, P₄S₇, PC1₅, Sb₂O₅, As₂O₅, BiOC1₂, SeOC1₂, SO₂C1₂, Sb₂S₅, VOC1₂, SiC1₄ and CrO₂Cl₂) dissolved in pyridine or 2,2,4-trimethyl pentane, to enhance subsequent staining of tissue components with toluidine blue, phosphotungstic acid-hematoxylin (PTAH), leukofuchsin, and dihydroxydinaphthyl-disulfide (DDD) was studied. Eight of these compounds were also tested for ability to enhance staining with Alcian blue 8GN and Luxol fast blue MBS. Nine of the 17 compounds produced increased staining of certain tissue components with leukofuchsin, 13 with toluidine blue, 16 with PTAH, and 16 with DDD. The results suggest additional approaches to identification of tissue entities by induced metachromatic basophilia and leukofuchsin positivity as well as by the other stains studied, and also suggest a number of hitherto unstudied modes of reaction between the dyes used and reactive groups of tissue components. Many reactions of the compounds tested, with reactive groups known to be present in tissue components, are basecatalyzed, so that choice of solvent can influence the results obtained.     

Identifying Monoaminergic,GABAergic, and Cholinergic Characteristics in Immortalized Neuronal Cell Lines

We measured the concentration of neurotransmitters in immortalized neural cell lines of hippo-campal, septal, brainstem and cerebellar origin. While in most of the cell lines, concentrations of monoamines, -aminobutyric acid (GABA) and acetylcholine were low, in some they were markedly higher. This made it quite easy to identify possible monoaminergic, GABAergic or cholinergic cell lines. However all the cell lines contained glutamate and aspartate and there were no outstanding differences in levels of these amino acids differences between the cell lines. Deprivation of serum, which made the cells acquire a more differentiated morphology, caused an increase in the intracellular concentrations of some compounds and a switch from multiple to a single transmitter in the case of some cell lines. It suggested that measurement of transmitter concentrations combined with serum deprivation studies, may provide an indication of the neurochemical characteristics of immortalised neuronal cell lines.     

Fluoride profiles in the cementum and root dentine of human permanent anterior teeth extracted from adult residents in a naturally fluoridated and a non-fluoridated area

Objectives: To determine the effect of water fluoride concentration on the fluoride profile across the entire thickness of the cementum and root dentine of human permanent anterior teeth in adults. Subjects: Twenty-eight human permanent anterior teeth from individuals aged from 30 to over 60 years were studied. Setting: Teeth were obtained from a natural high-fluoride area (West Hartlepool, UK; 1.0–1.3 ppm F in drinking water, WHP) and the other from a non-fluoridated naturally low fluoride area (Leeds, UK; 0.1 ppm F in drinking water, LDS). Design: Cementum and root dentine were sampled using an abrasive micro-sampling technique from the cementum surface to the pulpal surface of root dentine. Results: Fluoride concentration was higher in tooth roots (the cementum and dentine) taken from the naturally fluoridated area (WHP) than from the non-fluoridated area (LDS). Age and average fluoride concentration showed a positive correlation in WHP dentine, middle region of the root (r = 0.78, P < 0.001) and in the apical region of the root (r = 0.67, P < 0.05). WHP cementum had the strongest fluoride concentration correlation with age in the cervical region of the root (r = 0.67, P < 0.01). An analysis of variance (ANOVA) showed that the area (water fluoride content), age and number of years lived in the area combined with total age were significant. Conclusions : The fluoride content of cementum and root dentine in adult residents is related to fluoride content in drinking water.     

Fluorescence and Electron Microscopic Localization of F-actin in the Ependymocytes

The organization of F-actin in the ventricular system has been reported to display pronounced regional differences with respect to shape, size, and development. However, the real roles played by F-actin in these cells cannot be understood unless the precise localization of F-actin is defined. In the present study, we used double-fluorescence labeling to further examine the localization of F-actin in the ependymocytes and its spatial relation to the other two cytoskeletal components, microtubules and intermediate filaments. Then we converted fluorescence signals for F-actin to peroxidase/DAB reaction products by use of a phalloidin-based FITC-anti-FITC system. This detection technique provided an overview of the distribution of F-actin in the ependymocytes at the ultrastructural level, and has been proven to be helpful in correlating light and electron microscopic investigations. (J Histochem Cytochem 57:741–751, 2009)     

爪哇根结线虫Mi-毒性和无毒近等基因系的制备及其遗传变异的初步分析

爪哇根结线虫是以孤雌生殖方式繁殖的农作物重要病原物,通过将爪哇根结线虫-无毒群体在抗病番茄品种Momotaro上连续选择19代,获得了一对针对抗性基因Mi的毒性和无毒近等基因系,用102种引物对该近等基因系进行随机扩增多态性DNA（RAPD）分析显示,除一种引物外两者的RAPD带谱基本一致,证实了该毒性和无毒群体的基因组组成十分近似,对一种引物扩增出的一个无毒群体特异性的RAPD片段进行了克隆,Southern杂交结果提示该多态性片段及其同源序列在毒性选择过程中被从毒性线虫的基因组中删除,但DNA序列分析和数据库检索表明,该片段与迄今发表的核酸序列均不存在显著的同源性,因此尚无法预测其潜在的功能性,爪哇根结线虫Mi-毒性和无毒近等基因系的制备为进一步鉴定和分离该线虫毒性相关基因打下了坚实的基础。     

Application of Stains-All for Demarcation of Cement Lines in Methacrylate Embedded Bone

Cement lines provide a record of sites of past remodeling buried in the matrix of bone. A method is reported for application of Stains-all, a cationic carbocyanine dye, for demarcation of cement lines in bone. The method, which is simple, works well for both glycol methacrylate and methyl methacrylate undemineralized embedments and produces good concomitant staining of cytoplasm and nuclei of osteoblasts, osteoclasts and marrow cells.