Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.      

The expression of p120ctn protein in breast cancer is independent of alpha- and beta-catenin and E-cadherin.

Several studies have reported loss or alteration of expression of E-cadherin in breast cancer and more recently changes in levels of expression of the catenins. We used immunofluorescence to examine E-cadherin, alpha-catenin, beta-catenin, and p120ctn (formerly p120CAS) expression in 91 cases of invasive ductal carcinoma. As expected, all four proteins co-localize to the junctional regions of the cells. Although nuclear localization has been described for beta-catenin in colonic polyps, no examples were found in these breast cancer cases. We found that, although alteration is common in the catenins and E-cadherin, complete loss, as exemplified by E-cadherin in lobular carcinoma (where E-cadherin is frequently mutated), is rarely seen. In contrast, the catenin-related protein p120ctn shows an expression pattern that is significantly unrelated to the other catenins (or E-cadherin), including complete loss of expression in approximately 10% of the cases. No statistically significant correlations with traditional prognostic indicators were observed with any of these proteins. We conclude 1) that expression of E-cadherin and alpha- and beta-catenin are generally retained at the membrane although frequently reduced or altered, 2) that complete loss of p120ctn expression is seen in approximately 10% of the cases, and 3) that there is a significant correlation in the expression of E-cadherin and the catenins but no correlation between these molecules and p120ctn, suggesting an absence of coordinate regulation.     

Down syndrome and trisomy 18 in the bedouins

Direct chromosome preparations of neonatal cord blood provides the unique opportunity for rapid chromosome analysis (turnaround time; 6 hr), without the necessity of bone marrow aspiration. Based on 42 samples we confirm the finding of Garnham and Sutherland [1987] for suitability of cord blood for direct chromosome preparation. Procedural modifications are provided for higher yield of cells for chromosome analysis. The procedure may well be of major significance for rapid diagnosis of neonates who suffer from aneusomy.     

Alteration of pulmonary neuroendocrine cells during epithelial repair of naphthalene-induced airway injury

Whole-mount airway preparations isolated from the lungs of mice treated by intraperitoneal injection of naphthalene and allowed to recover for 5 days were examined for the distribution and abundance of solitary pulmonary neuroendocrine cells (PNECs) and neuroepithelial bodies (NEBs) along the main axial pathway of the right middle lobe. Sham mice treated with corn oil vehicle were examined in a similar manner. An antibody to calcitonin gene-related peptide, a neuroendocrine cell marker, was used to identify the location, size, and number of PNECs and NEBs in the airways. After naphthalene treatment and epithelial repair, NEBs were significantly increased along the walls of the airways as well as on branch point ridges. The surface area covered by NEBs composed of 20 or fewer PNECs was significantly enlarged after naphthalene treatment compared with control NEBs of an equivalent cell number. The PNEC number per square millimeter was also increased more than threefold above control values after naphthalene treatment. These findings provide further support for a key role of neuroendocrine cells in the reparative process of airway epithelial cell renewal after injury.     

Studies of efferent lymph cells from nodes stimulated with oxazolone.

Efferent lymph from nodes regional to areas of skin that had been treated with solutions of oxazolone in acetone was collected from unanaesthetized sheep. The application of 5% solutions of oxazolone to unsensitized sheep caused no signs of acute inflammation or ''shut-down'' of lymphocyte traffic; none the less, normal immune responses ensued so that immunoblasts, some containing immunoglobulin, were discharged into the lymph together with specific humoral antibodies. When previously sensitized sheep were challenged with 2.5% solutions of oxazolone the vigorous secondary responses were heralded by Arthus reactions, induced presumably by pre-existing antibodies, which were mainly of the IgG class. A similar sequence of events occurred in a thymus-deprived sheep which had undergone intra-uterine thymectomy at 60 days of gestation. Repeated applications of oxazolone to normal sheep did not exhaust or inhibit the characteristic changes in the flow and composition of the lymph. When immunoblasts from efferent lymph were radiolabeled with 125I-UdR and returned intravenously to the sheep they showed no significant tendency to localize either specifically or non-specifically in areas of skin that had been treated with contact-sensitizing chemicals.     

Comparison of frequency, intensity, and duration of aggressive responses in rats

Three judges were trained to evaluate the frequency, intensity, and duration of aggressive responses in rats. Intrajudge and interjudge reliability ranged from .84 to .99. Correlation coefficients between frequency, intensity, and duration of aggressive responses ranged from .89 to .99. All three measures were found to be highly reliable methods of measuring aggression.     

Analysis of variability of clinical manifestations in Waardenburg syndrome

Expression of clinical findings of Waardenburg syndrome type 1 (WS1) and type 2 (WS2) is extremely variable. Using our collection of 26 WS1 and 8 WS2 families, we analyzed the occurrence, severity, and symmetry of clinical manifestations associated with WS. We found significant differences between WS1 and WS2 in deafness, and in pigmentary and craniofacial anomalies. Factor analysis was used to identify manifestations which covaried, resulting in 2 orthogonal factors. Since mean factor scores were found to differ when compared between WS1 and WS2, we suggest that these factors could be useful in distinguishing WS types. We found that the WS gene was transmitted from mothers more often than from fathers. We also extensively examined the W-Index, a continuous measure of dystopia canthorum. Our data suggest that use of the W-Index to discriminate between affected WS1 and WS2 individuals may be problematic since 1) ranges of W-Index scores of affected and unaffected individuals over-lapped considerably within both WS1 and WS2, and 2) a considerable number of both affected and unaffected WS2 individuals exhibited W-index scores consistent with dystopia canthorum. Misclassification of families may have implications for risk assessment of deafness, since WS2 families have been reported to have greater incidence of deafness, as confirmed in our study. © 1995 Wiley-Liss, Inc.