Rob M.W. de Waal

Heterozygous Germline Mutations in the p53 Homolog p63 Are the Cause of EEC Syndrome

EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.

Expression pattern of apoptosis-related markers in Huntington's disease

Inappropriate apoptosis has been implicated in the mechanism of neuronal death in Huntington’s disease (HD). In this study, we report the expression of apoptotic markers in HD caudate nucleus (grades 1–4) and compare this with controls without neurological disease. Terminal transferase-mediated biotinylated-UTP nick end-labeling (TUNEL)-positive cells were detected in both control and HD brains. However, typical apoptotic cells were present only in HD, especially in grade 3 and 4 specimens. Expression of the pro-apoptotic protein Bax was increased in HD brains compared to controls, demonstrating a cytoplasmic expression pattern in predominantly shrunken and dark neurons, which were most frequently seen in grades 2 and 3. Control brains displayed weak perinuclear expression of the anti-apoptotic protein Bcl-2, whereas in HD brains Bcl-2 immunoreactivity was markedly enhanced, especially in severely affected grade 4 brains, and was observed in both healthy neurons and dark neurons. Caspase-3, an executioner protease, was only found in four HD brains of different grades and was not expressed in controls. A strong neuronal and glial expression of poly(ADP-ribose) polymerase (PARP)-immunoreactivity was observed in HD brains. These data strongly suggest the involvement of apoptosis in HD. The exact apoptotic pathway occurring in HD neurodegeneration remains yet unclear. However, the presence of late apoptotic events, such as enhanced PARP expression and many TUNEL-positive cells accompanied with weak caspase-3 immunoreactivity in severely affected HD brains, suggests that caspase-mediated neuronal death only plays a minor role in HD.

3-Nitropropionic acid induces cell death and mitochondrial dysfunction in rat corticostriatal slice cultures.

Exposure of organotypic rat corticostriatal slice cultures to the mitochondrial toxin 3-nitropropionic acid (3-NP) resulted in concentration-dependent loss of cresylviolet-stained cells and increase of lactate dehydrogenase and lactate efflux into the culture medium, indicators for cell death and metabolic activity in the slices, respectively. The involvement of apoptosis in these slices was suggested by using the terminal transferase-mediated biotinylated-UTP nick end-labeling (TUNEL) technique, and immunohistochemistry for the apoptosis-related markers Bax and Bcl-2. In 3-NP-exposed slices, TUNEL-positive cells were observed in both the striatum and the cortex but in different forms: striatal neurons were either diffusely stained or showed nuclear fragmentation, cortical neurons only exhibiting nuclear fragmentation. In 3-NP-exposed slices, the pro-apoptotic protein Bax was abundantly expressed, whereas the anti-apoptotic protein Bcl-2 was not expressed in striatal neurons. We suggest that both apoptosis and necrosis are involved in the 3-NP-treated slices, apoptosis as well as necrosis in the striatum and apoptosis in the cortex.

Immunohistochemical identification of Langerhans cells in normal epithelium and in epithelial lesions of the uterine cervix

Using the double label indirect immunofluorescence technique we have studied vimentin-positive cells present in normal ecto- and endocervical epithelium, subcolumnar reserve cell hyperplasia, and squamous metaplastic and dysplastic epithelium of the uterine cervix. Monoclonal antibodies to Ia- and T6-antigens were applied in the examination of the expression of these membrane markers by such cells. Our studies reveal the presence of a relatively large number of vimentin-positive and T6-positive (Langerhans) cells in normal ectocervical stratified squamous epithelium, a small number in endocervical columnar epithelium, and a larger number in subcolumnar reserve cell hyperplasia and in immature squamous metaplasia. In this respect, mature squamous metaplastic epithelium shows a great resemblance to normal ectocervical stratified squamous epithelium. In contrast with previous reports in the literature we could only demonstrate small numbers of Langerhans cells in cases of dysplasia. The clinicopathological significance of these findings is discussed.

Creatine protects against 3-nitropropionic acid-induced cell death in murine corticostriatal slice cultures.

In murine corticostriatal slice cultures, we studied the protective effects of the bioenergetic compound creatine on neuronal cell death induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). 3-NP caused a dose-dependent neuronal degeneration accompanied by an increased lactate dehydrogenase (LDH) activity in the cell culture medium. An increased ratio of lactate to pyruvate concentration in the medium suggested that metabolic activity shifted to anaerobic energy metabolism. These effects were predominantly observed in the 24-h recovery period after 3-NP exposure. Creatine protected against 3-NP neurotoxicity: LDH activity was reduced and aerobic respiration of pyruvate was stimulated, which resulted in lower lactate levels and less cell death. In both striatum and cortex, apoptosis in 3-NP-exposed slices was demonstrated by increased activation of the pro-apoptotic protein caspase-3 and by numerous cells exhibiting DNA fragmentation detected by the terminal transferase-mediated biotinylated-UTP nick end-labeling (TUNEL) technique. Creatine administration to the 3-NP-exposed corticostriatal slices resulted in a reduced number of TUNEL-positive cells in the recovery period. However, in the striatum, an unexpected increase of both TUNEL-positive cells and caspase-3-immunostained cells was observed in the exposure phase in the presence of creatine. In the recovery phase, caspase-3-immunostaining decreased to basal levels in both striatum and cortex. These findings suggest that 3-NP-induced neuronal degeneration in corticostriatal slices results from apoptosis that in the cortex can be prevented by creatine, while in the more vulnerable striatal cells it may lead to an accelerated and increased execution of apoptotic cell death, preventing further necrosis-related damage in this region.

Uncovering students' misconceptions by assessment of their written questions.

BackgroundMisconceptions are ideas that are inconsistent with current scientific views. They are difficult to detect and refractory to change. Misconceptions can negatively influence how new concepts in science are learned, but are rarely measured in biomedical courses. Early identification of misconceptions is of critical relevance for effective teaching, but presents a difficult task for teachers as they tend to either over- or underestimate students’ prior knowledge. A systematic appreciation of the existing misconceptions is desirable. This explorative study was performed to determine whether written questions generated by students can be used to uncover their misconceptions.MethodsDuring a small-group work (SGW) session on Tumour Pathology in a (bio)medical bachelor course on General Pathology, students were asked to write down a question about the topic. This concerned a deepening question on disease mechanisms and not mere factual knowledge. Three independent expert pathologists determined whether the content of the questions was compatible with a misconception. Consensus was reached in all cases. Study outcomes were to determine whether misconceptions can be identified in students’ written questions, and if so, to measure the frequency of misconceptions that can be encountered, and finally, to determine if the presence of such misconceptions is negatively associated with the students’ course formal examination score. A subgroup analysis was performed according to gender and discipline.ResultsA total of 242 students participated in the SGW sessions, of whom 221 (91xa0%) formulated a question. Thirty-six questions did not meet the inclusion criteria. Of the 185 questions rated, 11xa0% (nu2009=u200920) was compatible with a misconception. Misconceptions were only found in medical students’ questions, not in biomedical science students’ questions. Formal examination score on Tumour Pathology was 5.0 (SD 2.0) in the group with misconceptions and 6.7 (SD 2.4) in the group without misconceptions (pu2009=u20090.003).ConclusionsThis study demonstrates that misconceptions can be uncovered in students’ written questions. The occurrence of these misconceptions was negatively associated with the formal examination score. Identification of misconceptions creates an opportunity to repair them during the remaining course sessions, in advance of the formal examination.

BINDING OF THE AB43 PEPTIDE TO APOLIPOPROTEIN E AND ITS ROLE IN CLEARANCE

previously associated with AD including tau, apolipoprotein E, complement C1q, neuroligin and heat shock proteins. Studies are ongoing to determine which proteins have altered expression in AD tissue from patients at early versus late stages of disease and tissue from normal age-matched controls. Conclusions: The combined use of laser capture microdissection and LCMS to characterize proteins in specific brain structures or cell types has great promise for future studies. Future results using this technique will complement those from GWAS studies, potentially identifying protein pathways uniquely expressed or altered in AD. The function of these identified proteins and consequences of expression in AD can then be pursued in further research and could provide novel therapeutic targets.

THE HCMEC/D3 CELL LINE IS NOT SUITABLE AS A MODEL FOR Aβ TRANSPORT BY THE HUMAN BLOOD-BRAIN BARRIER

A MODEL FOR Ab TRANSPORT BY THE HUMAN BLOOD-BRAIN BARRIER Elisanne A.L.M. Biemans, H. Bea Kuiperij, Rob M.W. De Waal, Marcel M. Verbeek, Department of Neurology, Donders Centre for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands; 2 Department of Pathology, Donders Centre for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands. Contact e-mail: elisanne.biemans@ radboudumc.nl