Androgenetic/biparental mosaicism causes placental mesenchymal dysplasia.

BACKGROUND: Placental mesenchymal dysplasia (PMD) is a distinct syndrome of unknown aetiology that is associated with significant fetal morbidity and mortality. Intrauterine growth restriction is common, yet, paradoxically, many of the associated fetuses/newborns have been diagnosed with Beckwith-Wiedemann syndrome (BWS). METHODS: We report two cases of PMD with high levels of androgenetic (complete paternal uniparental isodisomy) cells in the placenta and document, in one case, a likely androgenetic contribution to the fetus as well. RESULTS: The same haploid paternal complement found in the androgenetic cells was present in coexisting biparental cells, suggesting origin from a single fertilisation event. CONCLUSIONS: Preferential allocation of the normal cells into the trophoblast explains the absence of trophoblast overgrowth, a key feature of this syndrome. Interestingly, the distribution of androgenetic cells appears to differ from that reported for artificially created androgenetic mouse chimeras. Androgenetic mosaicism for the first time provides an aetiology for PMD, and may be a novel mechanism for BWS and unexplained intrauterine growth restriction.

Intra- and intermolecular triplex DNA formation in the murine c-myb proto-oncogene promoter are inhibited by mithramycin.

Mithramycin inhibits transcription by binding to G/C-rich sequences, thereby preventing regulatory protein binding. However, it is also possible that mithramycin inhibits gene expression by preventing intramolecular triplex DNA assembly. We tested this hypothesis using the DNA triplex adopted by the murine c-myb proto-oncogene. The 5'-regulatory region of c-myb contains two polypurine:polypyrimidine tracts with imperfect mirror symmetry, which are highly conserved in the murine and human c-myb sequences. The DNA binding drugs mithramycin and distamycin bind to one of these regions as determined by DNase I protection assay. Gel mobility shift assays, nuclease and chemical hypersensitivity and 2D-gel topological analyses as well as triplex-specific antibody binding studies confirmed the formation of purine*purine:pyrimidine inter- and pyrimidine*purine:pyrimidine intra-molecular triplex structures in this sequence. Mithramycin binding within the triplex target site displaces the major groove-bound oligonucleotide, and also abrogates the supercoil-dependent H-DNA formation, whereas distamycin binding had no such effects. Molecular modeling studies further support these observations. Triplex-specific antibody staining of cells pretreated with mithramycin demonstrates a reversal of chromosomal triplex structures compared to the non-treated and distamycin-treated cells. These observations suggest that DNA minor groove-binding drugs interfere with gene expression by precluding intramolecular triplex formation, as well as by physically preventing regulatory protein binding.

In situ analysis of the transcriptional activity of integrated viral DNA using tyramide-FISH.

Infection by the oncogenic human papillomavirus (HPV) types 16 and type 18 can progress to cancers. Two well studied cervical carcinoma cell lines, SiHa and CaSki, contain two to four copies, or several hundred copies of integrated HPV-16, respectively. To define the chromosomal loci from which HPV mRNAs are transcribed in these cells, we have simultaneously visualized chromosomal DNA territories, HPV DNA or nascent HPV RNA sequences by using a highly sensitive in situ hybridization (T-FISH) technique employing deposition of fluorescent tyramides. We found that, in SiHa cells, nascent HPV RNAs co-localized with both integrated HPV copies on chromosome 13. Surprisingly, in CaSki cells, nascent HPV RNA only co-localized with one minor HPV DNA-positive locus on chromosome 14. The DNA signal intensity of this locus was consistent with a single to a few HPV intergrants. The tyramide methodologies described here provide an in-depth molecular cytological analyses applicable to research and diagnosis.

The value of CAGE, CUGE, and AUDIT in screening for alcohol abuse and dependence among college freshmen.

BACKGROUND: This study attempted to (1) determine the prevalence of alcohol problems in college freshmen, (2) assess the performance of both the CAGE and the Alcohol Use Disorders Identification Test (AUDIT) questionnaires in this population, and (3) assess the possibility of improving the CAGE and/or AUDIT. METHODS: A sample of 3564 consecutive college freshmen, with a mean age of 18 years, at the Catholic University of Leuven, (Belgium) completed, during a cross-sectional study, a questionnaire assessing drinking behavior and identifying students at risk as defined by DSM-IV criteria. The questionnaire also included the CAGE questionnaire and the AUDIT. Calculations of sensitivity, specificity, negative predictive value, positive predictive value, likelihood ratios, and receiver operating characteristic curves for different scores of the CAGE and the AUDIT were performed, using DSM-IV criteria as the reference standard. RESULTS: The area under the receiver operating characteristic curve of the CAGE and the AUDIT was 0.76 and 0.85, respectively. The cutoff score of 1 for the CAGE was associated with a sensitivity of 42%, a specificity of 87%, a positive predictive value of 36%, and a negative predictive value of 90%. A score of 6 or more for the AUDIT gave a sensitivity of 80%, a specificity of 78%, a positive predictive value of 37%, and a negative predictive value of 77%. These results were related with a prevalence of 14.1% of alcohol problems. Replacing one question of the CAGE by "often driving under the influence" resulted in the CUGE (acronym for "cut down, under influence, guilty feelings, and eye opener"), with an area under the curve of 0.96, a positive likelihood ratio of 8.7, and a negative likelihood ratio of 0.04. CONCLUSIONS: Prevalence of alcohol problems in college students is confirmed to be high. When screening for alcohol problems in a college freshmen population, one question seems extremely important. The newly constructed CUGE questionnaire may improve screening efforts in students, compared with existing questionnaires.

Localization of HuC (ELAVL3) to chromosome 19p13.2 by fluorescence in situ hybridization utilizing a novel tyramide labeling technique.

HuC is a neural-specific member of the Elav family of RNA-binding proteins. This highly conserved gene family plays a crucial role in neurogenesis, and HuC (HGMW-approved symbol ELAVL3) is expressed at an early stage of neural development. Using a novel tyramide fluorescence in situ hybridization (T-FISH) technique, we localized HuC to chromosome 19p13.2. This localization was confirmed by radiation hybrid mapping and coincides with that of HuR (HGMW-approved symbol ELAVL1), another elav family member. Dual T-FISH analysis with HuC and HuR probes, however, indicated distinct loci, with HuC being centromeric to HuR. This study demonstrates the utility of T-FISH in colocalizing two genes on the same chromosomal preparation using only biotinylated probes.

Localization of human elav-like neuronal protein 1 (Hel-N1) on chromosome 9p21 by chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization.

Hel-N1 is a member of the highly conserved elav family of neuronal genes. It shares considerable sequence homology with HuD, another human member, and both genes are expressed in brain. HuD was recently mapped to chromosome 1p34. Here, we have utilized chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization to map Hel-N1 to chromosome 9p21. The different chromosomal locations of these homologous genes underscore their distinct identities.

Adolescents' attitude towards carrier testing for cystic fibrosis and its relative stability over time.

Attitudes towards cystic fibrosis (CF) carrier testing, benefits of and barriers to having such a test were assessed within a randomly selected group of high school students in Flanders, after they had received sufficient basic information about the nature and the mode of inheritance of CF. Attitudes towards carrier testing for CF were not negative, but the majority preferred to wait to have a test. This result changed little after 6 months. A hypothetical testing offer from the Medical School Health Service elicited positive answers from nearly two thirds, suggesting that such an offer may function as a cue to action. Nevertheless, the appropriateness of such an offer may be questioned, considering the disadvantages of testing adolescents. Concern about a negative impact of the carrier status on self-image was reported by 10% of the students. These findings suggest that education about genetics is not only a prerequisite for allowing more informed decisions about CF carrier testing, but also for avoiding negative psychosocial effects of such a test.

Cell-type and amyloid precursor protein-type specific inhibition of A beta release by bafilomycin A1, a selective inhibitor of vacuolar ATPases.

Treatment of human 293 cells transfected with amyloid precursor protein (APP)K595N,M596L (the "Swedish" mutation) with a specific inhibitor of the vacuolar H(+)-ATPases, bafilomycin A1 (baf A), leads to a potent inhibition of the release of the A beta peptide. This is accompanied by a selective inhibition of beta-secretase activity. Surprisingly, baf A did not inhibit the production of A beta from either wild-type APP (WT APP) or from APPv7171 (the "Hardy" mutation), expressed in the same cell type. In contrast, the robust production of A beta from a human neuroglioma-derived cell line (HS683) transfected with WT APP, or from primary human mixed brain cultures (HMBC) expressing genomic WT APP, were also effectively inhibited by baf A. The inhibition of A beta production from the HMBC was also accompanied by the inhibition of beta-s-APP release. No inhibition of alpha-s-APP release was seen in any of the cell types tested. These results indicate that intracellular acidic processes are rate-limiting for beta-secretase cleavage and A beta production from SW APP, but not WT APP, in the peripheral 293 cell line. Furthermore, such acidic processes also play a rate-limiting role in A beta release from human central nervous system-derived cells, including HMBC. Differential trafficking of the SW APP into an acidic compartment conducive to beta-secretase cleavage and A beta release could be one explanation for the increased production of A beta observed on expression of this mutation.

Evidence for a nonsecretory, acidic degradation pathway for amyloid precursor protein in 293 cells. Identification of a novel, 22-kDa, beta-peptide-containing intermediate.

We have analyzed the metabolic pathway of maturation of APP751 in stably transfected 293 cells, in the presence of either of the cysteine protease inhibitors leupeptin or E-64. Metabolic labeling, followed by immunoprecipitation at various times in the chase with a rabbit polyclonal antibody (anti-BX6) specific to the carboxyl-terminal end of amyloid precursor protein (APP), revealed the accumulation of a novel approximately 22-kDa carboxyl-terminal fragment (22-CTF) in the inhibitor-treated cells. This fragment, which was not detectable in untreated cells, was immunoprecipitated by four separate antibodies to the carboxyl-terminal region of APP as well as by polyclonal and monoclonal antibodies specific to the first 16 amino acids of the beta-peptide domain. Antibodies to the amino-terminal end of APP do not, however, recognize the fragment. Co-treatment of the inhibitor-treated cells with either of the lysosomotropic agents chloroquine or ammonium chloride completely blocked the generation of this fragment but did not significantly affect APP maturation or secretion. All, however, slowed the intracellular turnover of the cell-associated, approximately 9-kDa carboxyl-terminal fragment (c-CTF) produced during constitutive secretion. Densitometric analyses of these results suggest that this non-secretory pathway of APP degradation, mediated by cysteine proteases in an intracellular acidic compartment, accounts for approximately 70% of total APP metabolism and that a key processing intermediate in this pathway is a 22-kDa, beta-peptide-containing APP carboxyl-terminal fragment. It is possible that inefficient degradation of such an intermediate leads to the formation of aggregating beta-peptide.

Overexpression of tau in a nonneuronal cell induces long cellular processes.

The ways in which the various microtubule-associated proteins (MAPs) contribute to cellular function are unknown beyond the ability of these proteins to modify microtubule dynamics. One member of the MAP family, tau protein, is restricted in its distribution to the axonal compartment of neurons, and has therefore prompted studies that attempt to relate tau function to the generation or maintenance of this structure. Sf9 cells from a moth ovary, when infected with a baculovirus containing a tau cDNA insert, elaborate very long processes. This single gene product expressed in a foreign host cell grossly alters the normal rounded morphology of these cells. The slender, relatively nonbranched appearance of these processes as well as their uniform caliber resembles the light-microscopic appearance of axons observed in several neuronal culture systems. Immunolabeling of the tau-expressing Sf9 cells demonstrated tau reactivity in the induced processes, and EM that microtubule bundles were present in the processes. Microtubule stabilization alone was insufficient to generate processes, since taxol treatment did not alter the overall cell shape, despite the induction of microtubule bundling within the cell body.