Detection of trisomy 8 in hematological disorders by in situ hybridization

An alphoid repetitive DNA (D8Z2) probe specific for the pericentromeric region of chromosome 8 was used to detect extra copies of chromosome 8 in bone marrow cells obtained from 10 patients with hematological disorders and five controls. Numerical aberrations of chromosome 8 were established by conventional banding techniques. Trisomy 8 was found in four patients with myelodysplastic syndrome (MDS) and three with acute myeloid leukemia (AML). Three additional patients with MDS exhibited an extra chromosome 8 in only one metaphase. In five of the seven trisomy cases, the presence of the trisomy 8 clone was confirmed by in situ hybridization (ISH). In one case of AML with trisomy 8, detected by GTG-banding, no significant numbers of cells containing three spots were found using the alphoid repetitive probe; however, hybridization with a chromosome 8-specific library revealed that the alleged extra chromosome 8 was a translocation chromosome containing only the long arm of chromosome 8. Due to a lack of material, it was not possible to achieve optimal ISH results on the trisomy 8 bone marrow cells of patient 7. In the three MDS patients with a single trisomy 8 metaphase, a slight, albeit significant, increase of trisomy 8 interphase cells was found with ISH. We conclude that this probe is useful for cytogenetic studies. Moreover, ISH, in general, is a powerful tool for precise classification of chromosomal aberrations and can also contribute significantly to the clinical evaluation of patients with hematological disorders.     

Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune.

Complementary DNA was synthesized on polyadenylated RNA from a dikaryotic mycelium of the basidiomycete Schizophyllum commune bearing fruiting body initials. The complementary DNA was cloned into the PstI site of pBR327 by the deoxyguanidylate-deoxycytidylate tailing approach. After transformation into Escherichia coli cells, a differential screening was performed by colony hybridization with complementary [32P]DNA made on the RNAs of the monokaryon and dikaryon strains. Two clones were selected for further analysis by Northern blotting and hybrid release translation. Clone 1D10 hybridized with an mRNA of 775 nucleotides, coding for a polypeptide with an Mr of 15,000. Although this RNA was present in both monokaryotic and dikaryotic mycelia, its concentration appeared to change considerably over time and with different cultivation conditions. This mRNA is probably the most abundantly expressed sequence in S. commune. Clone 1G2 and its homologs hybridized with an mRNA of 650 nucleotides, coding for a polypeptide with an Mr of 13,000. This gene was exclusively expressed in the dikaryon strain. In liquid-grown cultures, the concentration of this mRNA was low but increased ca. 20-fold during the establishment of fruiting body primordia. A chromosomal fragment of 9 kilobase pairs which contained the 1G2 gene was cloned into pBR327 and used as a probe in Northern blot hybridization. It was found that surrounding sequences were not expressed at the same time or to the same extent as the 1G2 gene.     

Ultrastructural differences between wall apices of growing and non-growing hyphae ofSchizophyllum commune

Summary Newly synthesized chitin at the hyphal apex ofSchizophyllum commune was shown to be highly susceptible to chitinase degradation and solubilization by dilute mineral acid. With time this chitin became gradually more resistant to these treatments. With a combination of the shadow-cast technique and electron microscopic autoradiography it could be shown that this process occurred as the newly synthesized chitin moved into subapical parts of growing hyphae but also in non-growing apices which had ceased growth after incorporation of theN-acetyl[6-³H]glucosamine. These results are in agreement with a model which explains apical morphogenesis by assuming that the newly synthesized wall material at the apex is plastic due to the presence of individual polymer chains but becomes rigidified because of subsequent physical and chemical changes involving these polymers.Dedicated to Dr. A.Quispel, Professor of Botany at the University of Leiden, on occasion of his retirement.     

A scoring system for coat and tail condition in ringtailed lemurs,Lemur catta

Coat condition can be influenced by a wide variety of disorders and thus provides a useful tool for noninvasive health and welfare assessments in wild and captive animals. Using Lemur catta as an exemplar, we offer a 6‐step scoring system for coat and tail condition, ranging from perfectly fluffy to half or more of body and tail being hairless. The categories are described in detail and illustrated with sample pictures from a wild population in Berenty Reserve, Madagascar. Furthermore, we elaborate on intermediate conditions and discoloration of fur. Coat condition scoring allows the comparison between years, seasons, and the effect of toxin, disease or stress. Although this system was developed for wild L. catta, we believe it can also be of value for other species. We recommend scoring coat condition in healthy wild mammal populations to give a baseline on yearly and seasonal variations vs. deteriorating health conditions or pathology. Am. J. Primatol. 71:183–190, 2009. © 2009 Wiley‐Liss, Inc.     

Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane–cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane–cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.     

Spectro-Temporal Weighting of Loudness

Real-world sounds like speech or traffic noise typically exhibit spectro-temporal variability because the energy in different spectral regions evolves differently as a sound unfolds in time. However, it is currently not well understood how the energy in different spectral and temporal portions contributes to loudness. This study investigated how listeners weight different temporal and spectral components of a sound when judging its overall loudness. Spectral weights were measured for the combination of three loudness-matched narrowband noises with different center frequencies. To measure temporal weights, 1,020-ms stimuli were presented, which randomly changed in level every 100 ms. Temporal weights were measured for each narrowband noise separately, and for a broadband noise containing the combination of the three noise bands. Finally, spectro-temporal weights were measured with stimuli where the level of the three narrowband noises randomly and independently changed every 100 ms. The data consistently showed that (i) the first 300 ms of the sounds had a greater influence on overall loudness perception than later temporal portions (primacy effect), and (ii) the lowest noise band contributed significantly more to overall loudness than the higher bands. The temporal weights did not differ between the three frequency bands. Notably, the spectral weights and temporal weights estimated from the conditions with only spectral or only temporal variability were very similar to the corresponding weights estimated in the spectro-temporal condition. The results indicate that the temporal and the spectral weighting of the loudness of a time-varying sound are independent processes. The spectral weights remain constant across time, and the temporal weights do not change across frequency. The results are discussed in the context of current loudness models.     

Co-occurrence analysis of insertional mutagenesis data reveals cooperating oncogenes

MOTIVATION: Cancers are caused by an accumulation of multiple independent mutations that collectively deregulate cellular pathways, e.g. such as those regulating cell division and cell-death. The publicly available Retroviral Tagged Cancer Gene Database (RTCGD) contains the data of many insertional mutagenesis screens, in which the virally induced mutations result in tumor formation in mice. The insertion loci therefore indicate the location of putative cancer genes. Additionally, the presence of multiple independent insertions within one tumor hints towards a cooperation between the insertionally mutated genes. In this study we focus on the detection of statistically significant co-mutations. RESULTS: We propose a two-dimensional Gaussian Kernel Convolution method (2DGKC), a computational technique that identifies the cooperating mutations in insertional mutagenesis data. We define the Common Co-occurrence of Insertions (CCI), signifying the co-mutations that are statistically significant across all different screens in the RTCGD. Significance estimates are made on multiple scales, and the results visualized in a scale space, thereby providing valuable extra information on the putative cooperation. The multidimensional analysis of the insertion data results in the discovery of 86 statistically significant co-mutations, indicating the presence of cooperating oncogenes that play a role in tumor development. Since oncogenes may cooperate with several members of a parallel pathway, we combined the co-occurrence data with gene family information to find significant cooperations between oncogenes and families of genes. We show, for instance, the interchangeable cooperation of Myc insertions with insertions in the Pim family. AVAILABILITY: A list of the resulting CCIs is available at: http://ict.ewi.tudelft.nl/~jeroen/CCI/CCI_list.txt.