A Method of Orienting Paraffin Sections for Three-Dimensional Reconstructions

To make reconstructions from serial sections, reference points are needed to orient the sections. Such points can be provided after paraffin embedding by cutting the bottom face of the block to form a plane and adding a groove along the center of this plane. The plane and groove are coated with Mimeograph Correction Fluid and the block is built up by dipping in hot paraffin so that the marked plane lies inside the block. Each section will have a blue line with a notch in it representing the plane and groove. This line remains through staining and is used to orient each section with respect to an eyepiece reticle. The reticle, in effect, supplies X and Y coordinates for every point in the specimen while the number of each section counted from one end is a Z coordinate.     

Polyaza crown ether as non-nucleosidic building blocks in DNA-conjugates

The synthesis of amphiphilic polyaza crown ether monomers X (palmityl-substituted), Y (cholesteryl-substituted) and Z (dipalmityl-subtituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis. Thermal denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y, and Z) with different lipophilicity showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer.     

Cusp size, sexual dimorphism, and heritability of cusp size in twins.

Overall measures of mandibular molars reflect the combined size contributions of the component cusps and ridges. Until now, the size hierarchy of primary and permanent mandibular molar cusps remained unclear. This paper utilizes the relative plane surface areas (basal area dimensions) of the individual molar cusps, as assays of cusp size to demonstrate cusp size variations within populations, antimere cuspal variations, sexual dimorphism, and, the heritability of cusp size. Duplicate dental casts from 199 pairs of like-sexed twins provide the raw dats. Defined anatomic landmarks on the occlusal surfaces were reduced to X-Y rectangular coordinates prior to the computation of the basal areas dimensions. The results establish a cusp size hierarchy specific for molar type, i.e., five-cusped molars with a distal fovea and distal marginal ridge (5fd), five-cusped molars without a distal fovea and without a distal marginal ridge (5o), and four-cusped molars (4c). Sexual dimorphism in cusp size is apparent in 5fd molar cusped but not in 5o molar cusps. However, males have a significantly higher frequency of 5fd molars. Females have a higher frequency of smaller 5o and 4c molars which have fewer crown components. Moreover, female 5o molars have cusps as large as or larger than 5o male molor cusps. Right-side-left-side differences exist between antimere cusps based on relatively low correlations. The mirroring of molor types occurs infrequently. When observed, most intrapair differences for cusp size, using F-ratios, indicate a low component of hereditary variability.     

Reproducibility of Frankfort Horizontal Plane on 3D Multi-Planar Reconstructed MR Images

Objective

The purpose of this study was to determine the accuracy and reliability of Frankfort horizontal plane identification using displays of multi-planar reconstructed MRI images, and propose it as a sufficiently stable and standardized reference plane for craniofacial structures.

Materials and Methods

MRI images of 43 subjects were obtained from the longitudinal population based cohort study SHIP-2 using a T1-weighted 3D sequence. Five examiners independently identified the three landmarks that form FH plane. Intra-examiner reproducibility and inter-examiner reliability, correlation coefficients (ICC), coefficient of variability and Bland-Altman plots were obtained for all landmarks coordinates to assess reproducibility. Intra-examiner reproducibility and inter-examiner reliability in terms of location and plane angulation were also assessed.

Results

Intra- and inter-examiner reliabilities for X, Y and Z coordinates of all three landmarks were excellent with ICC values ranging from 0.914 to 0.998. Differences among examiners were more in X and Z than in Y dimensions. The Bland–Altman analysis demonstrated excellent intra- as well as inter-examiner agreement between examiners in all coordinates for all landmarks. Intra-examiner reproducibility and inter-examiner reliability of the three landmarks in terms of distance showed mean differences between 1.3 to 2.9 mm, Mean differences in plane angulation were between 1.0° to 1.5° among examiners.

Conclusion

This study revealed excellent intra-examiner reproducibility and inter-examiner reliability of Frankfort Horizontal plane through 3D landmark identification in MRI. Sufficiently stable landmark-based reference plane could be used for different treatments and studies.     

Crown diameters of the deciduous teeth in Australian Aboriginals

Mesiodistal and buccolingual crown diameters were measured from dental casts representing the deciduous dentitions of 197 Aboriginal children from the Northern Territory of Australia. Double determination analysis indicated that the semi-automatic recording procedure used was reliable leading to observer errors of no practical significance. Tooth-size was greater in the male subjects but the sexual dimorphism was less marked than in the permanent teeth of the same subjects. The mandibular teeth were more uniform than maxillary with respect to buccolingual size relative to mesiodistal. Extremes of general tooth-size were more marked in the deciduous dentition than in the permanent as a consequence of the relatively large deciduous second molar which in Aboriginals approximates in size the permanent first molar of many other ethnic groups.     

Patterns of dental wear in the Lengua Indians of Paraguay

Wear patterns were examined on dental casts of 202 living Lengua Indians from the Chaco area of Paraguay. Consideration was given to the development of the molar helicoidal plane, age-related changes in occlusal attrition, coalescence of dentine exposures, interproximal attrition, and erupted crown height. This study lends support to Osborn's theory of the helicoidal plane development by showing that attrition enhances rather than modifies posteruption molar occlusal planes. The rate of interproximal attrition was found to slow down with the eruption and functional initiation of the third molars. Sinuous and cavo-convex interproximal contact areas that are generated with age, however, appeared to be less abrasion resistant than straight surfaces, hence leading to an increase in interproximal attrition rates with advanced age. Maximum crown height reduction occurred between the ages of 20 and 40 years in central incisors, canines, and first molars. Kruskal-Wallis tests and log linera models failed to demonstrate significant sexually dimorphic or antimeric differences in wear patterns of Lengua teeth.     

Semi-automated topographic mapping of fracture surfaces through stereo-photogrammetry

By equipping a standard Hilger-Watts stereo-photogrammetry viewer with transducers, sets of (X, Y, Z) coordinates corresponding to features within fracture surfaces can be obtained. Transducers mounted so as to monitor the the X and Y translations of the viewing tray, when incorporated with a third transducer to measure the rise and fall of the light spot used to measure apparent height changes, supply three voltage signals corresponding to the coordinates of any feature within the fracture surface. These signals are then processed through an analog/digital converter and the digitized signals stored on diskette on an ATT 6300 computer. The voltage files can then be converted to calibrated, normalized files of (X, Y, Z) coordinates using a menu-driven program. Inexpensive commercially available software is then incorporated to use this data to generate calibrated topographic maps. In this way, detailed contour maps, as well as carpet plots and line profiles, can be generated from any fracture surface.     

Contours of maxillary molars studied in Australian aboriginals

Distances from the central pit to the perimeter of the crown of permanent upper molars were measured on standardized occlusal photographs of dental casts representing 210 male and 181 female Aboriginals from Yuendumu in the Northern Territory of Australia. In both males and females the first molar was the largest tooth but it showed least variability. Variabilities of the distances tended to be greater for radii constructed in the buccolingual direction than for the transverse mesiodistal radii. The most marked size reduction in the molar series from first to third related to the distolingual part of the crown, which was also the most variable region. Size differences between molars in the mesial contour radii were not marked. Sexual dimorphism was evident in most crown radii, being most marked for the second molar.     

Holozygosity for sex-linked genes in males of the termiteIncisitermes schwarzi

The termite Incisitermes schwarzi has multiple sex chromosomes that have arisen by repeated translocations between autosomes and previously existing sex chromosomes. Two sex-linked allozyme loci--Acp-1 and Est-3--are holozygous, not hemizygous, in males (the heterogametic sex). Both loci show less than 1% crossing-over between X and Y chromosomes, and alleles of both are in marked disequilibrium with respect to X vs Y linkage. The two loci assort independently in female meiosis, indicating that they lie on different sex chromosomes. But they are tightly linked in male meiosis because of nonrandom assortment of the multiple X and Y chromosomes in males of this species. The findings of holozygosity and strong linkage disequilibrium suggest that differential selection in the two sexes at or near these loci may be responsible for the establishment of the translocations in this species. The existence of active Y-linked alleles also suggests that the translocations may have occurred recently.     

The location of highly repetitious DNA in the somatic chromosomes of Drosophila melanogaster

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirms earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.     

Fine mapping of the distal short arm of the human X chromosome using X/Y translocations.

The loci for steroid sulfatase (STS), the deficiency of which causes X-linked ichthyosis, the cell surface antigen 12E7 (MIC2X), and the blood group antigen Xg (Xg) have been mapped to Xp22.3. These loci are of particular interest since they do not appear to undergo X-chromosome inactivation. In an attempt to establish the relative order of STS and MIC2X, fibroblasts from carriers of four different X/Y translocations and an X/10 translocation were obtained and fused with mouse cell lines deficient in hypoxanthine phosphoribosyltransferase. The breakpoints on the X chromosome in these five translocations are in Xp22. Several independent clones from each fusion were isolated in HAT medium. The clones were examined cytogenetically, and in each case at least two independent clones were identified that have an active X/Y or X/10 translocation chromosome in the absence of other X or Y material. These clones were then tested for STS and 12E7 expression. In two of the X/Y translocations, the markers, STS and 12E7, were both absent. In the X/10 and a third X/Y translocation, both markers were retained. In each of three clones containing the fourth X/Y translocation, STS activity was retained but 12E7 antigenicity was lost. Assuming that this is a simple translocation and does not represent a more complex rearrangement, these results suggest that MIC2X is distal to STS.     

Restriction fragment length polymorphisms linked to genes for resistance to crown rust (Puccinia coronata) in near-isogenic lines of hexaploid oat (Avena sativa).

Crown rust, perhaps the most important fungal disease of oat, is caused by Puccinia coronata. An examination of near-isogenic lines (NILs) of hexaploid oat (Avena sativa) was conducted to identify markers linked to genes for resistance to crown rust. These lines were created such that a unique resistance gene is present in each of the two recurrent parent backgrounds. The six NILs of the current study, X434-II, X466-I, and Y345 (recurrent parent C237-89) and D486, D494, and D526 (recurrent parent Lang), thus provide a pair of lines to study each of three resistance genes. Restriction fragment length polymorphisms and resistance loci were mapped using BC1F2 populations. Three markers were found linked to a locus for resistance to crown rust race 203, the closest at 1.9 cM in line D494 and 3.8 cM in line X466-I. In lines D526 and Y345 a marker was placed 1.0 and 1.9 cM, respectively, from the locus conferring resistance to crown rust race 345, and in D486 and X434-II a marker mapped at 8.0 and 10.2 cM from the locus for resistance to rust race 264B.     

Sexual dimorphism in crown units of mandibular deciduous and permanent molars in Australian Aborigines.

Sexual differences in the crown units of mandibular molars were investigated in Australian Aborigines. The first and second deciduous molars (dm1 and dm2), and first to third permanent molars (M1, M2 and M3) were measured on dental casts using a sliding caliper. Measurements of tooth crowns included overall mesiodistal and buccolingual diameters, as well as the mesiodistal and buccolingual diameters of the trigonid and talonid. Percentage dimorphism values were greater in the talonid dimensions than the trigonid, indicating that sex differences tend to be larger in the later-developing crown units. Sex differences in mesiodistal diameters increased from dm1 to M2 but decreased for M3, the tooth that showed the least dimorphism of all the molars. This result seems to be due to the marked variability in size of the M3 between individuals.     

Variation of constitutive heterochromatin in the sex chromosomes of the rodent Bandicota bengalensis bengalensis (Gray)

Bandicota bengalensis bengalensis (Gray) trapped from different localities of India and Nepal exhibited a marked variation in the size and morphology of sex chromosomes. Three types of X's were found; A) simple acrocentric, B) composite subtelocentric and C) composite submetacentric X with their relative sizes 5.9%, 7.5% and 9.6% of the genome respectively. The autosomes remained unaltered. It was shown that this variation in the size of sex chromosomes was caused by deletion of constitutive heterochromatin. The Y chromosome was also found to be variable. Usually a large X was combined with a large Y. The preponderance of homozygotes for each type of X chromosome in populations, suggested the probable role of sex chromosomes heterochromatin in speciation.     

Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis

Cell surface molecules undergo specific changes during apoptosis, including the expression of phosphatidylserine (PS) and some proteins and alterations in sugar chains. Among the various sugar chains on the cell surface, Lewis X (Le(X)) and Lewis Y (Le(Y)) antigens are key determinants for a variety of biological processes. We studied the changes in Le(X) and Le(Y) expression in Jurkat cells, a human T cell line, during apoptosis. Flow cytometry showed that Le(X) and Le(Y) antigen expression was enhanced on the cell surface during apoptosis induced by anti-Fas antibody. To clarify the mechanism of enhanced Le(X) and Le(Y) expression, we assessed the expression levels of fucosyltransferase (FUT1, 2, 3-5-6, 4, and 9) mRNAs that are predominantly expressed in Jurkat cells and which are considered to form Le(X) and Le(Y). The expression of FUT4 mRNA was up-regulated after exposing cells to anti-Fas antibody. Moreover, the increase in Le(X) and Le(Y) antigen levels was significantly suppressed by caspase 3 or 8 inhibitors. These results indicated that the induction of FUT (mainly FUT4), the gene expression of which is mediated by signals downstream of caspase 3, increases Le(X) and Le(Y) expression in apoptotic cells.     

A technique for quantitating airway size from bronchial casts

To develop a technique for quantitating the size of airways at various positions in the bronchial tree, we analyzed casts of formalin-fixed excised lungs of five mature male ferrets. The left lower lobe of each cast was dissected, the diameter and position of each terminal bronchiole were entered into a computer programmed to reconstruct the airway system, and the cross-sectional areas of 120 conducting airways were measured. The fraction of the lobe served by each measured airway was estimated by dividing the sum of the squared diameters of the terminal bronchioles subtended by that airway by the summed squared diameters of all terminal bronchioles in the lobe. In each cast the relationship between an airway's cross-sectional area (Y) and the fraction of the lobe it was estimated to subtend (X) was described (0.91 less than R2 less than 0.95) by the expression ln(Y) = A + B ln(X) + C [ln(X)]2. Linear regression of ln(Y) on ln(X) for 30-50 airways estimated to serve fractions of the lobe around each of three arbitrarily selected levels (airways serving 0.7, 2.2, and 9.5% of the lobe) was adequate to characterize the area of airways at each level in each of the five animals with 95% confidence intervals narrower than 8% of the estimated area. Variability of airway size at each level among the five casts was modest, suggesting that this technique identified analagous airways in the various animals. Interindividual variability did not increase when the data were reanalyzed with terminal units defined on the basis of airway diameters rather than on the morphological identification of terminal bronchioles.     

Extracellular ATP facilitates flow-induced vasodilatation in rat small mesenteric arteries

ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 microM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 microM), a P2Y agonist, or 3'-O-(4-benzoyl)benzoyl ATP (BzATP; 10 microM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 microM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist alpha,beta-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 microM), a P2Y antagonist, and suramin (100 microM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 microM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5'-(3-thiotriphosphate)trilithium salt (1 microM) and adenosine 5'-(beta,gamma-imido) triphosphate tetralithium salt hydrate (10 microM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.     

Meiosis in the male mouse. An autoradiographic investigation

Meiosis in the male mouse has been studied autoradiographically in air-dried preparations. Information has been obtained on the relative rates of DNA synthesis and the lengths of the S-periods in spermatogonia and spermatocytes. The average rate of synthesis in the spermatocyte is lower, and the S-period is of longer duration than the preceding spermatogonial generations. The labelling pattern of the sex-chromosomes and autosomes observed at diakinesis and metaphase II in cells labelled at the spermatocyte S-period appears to be similar to that found in cells labelled during the spermatogonial S-periods. Replication in the autosomes commences before the sex-chromosomes. Late replicating autosomal centric regions show a marked degree of asynchrony in labelling both between and within bivalents. The Y chromosome starts and finishes replication later than the X. There is a short, late-replicating, segment of the X in the vicinity of the centromere. There is a short, early-replicating segment of the Y in the vicinity of the centromere which may represent the euchromatic short arm. The X and Y appear to associate at diakinesis by the distal ends of their long arms.