Elevated plasma cholesterol levels are considered responsible for excess cardiovascular morbidity and mortality. Cholesterol in plasma is tightly controlled by cholesterol within cells. Here, we developed and applied an integrative functional genomics strategy that allows systematic identification of regulators of cellular cholesterol levels. Candidate genes were identified by genome-wide gene-expression profiling of sterol-depleted cells and systematic literature queries. The role of these genes in cholesterol regulation was then tested by targeted siRNA knockdown experiments quantifying cellular cholesterol levels and the efficiency of low-density lipoprotein (LDL) uptake. With this strategy, 20 genes were identified as functional regulators of cellular cholesterol homeostasis. Of these, we describe TMEM97 as SREBP target gene that under sterol-depleted conditions localizes to endo-/lysosomal compartments and binds to LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). Taken together, TMEM97 and other factors described here are promising to yield further insights into how cells control cholesterol levels.

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A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

Several oncogenes isolated by the NIH/3T3 transformation assay, i.e., dbl, dbs, lbc, lfc, lsc, net, ost and tim, contain a Dbl homology (DH) and a pleckstrin-homology (PH) domain and act as GEFs (guanine nucleotide exchange factors) for Rho-like GTPases. In a search for genes with oncogenic potential in DNA from the monocytic leukaemia cell line U937, we identified an amino-terminal truncated form of gef-h1, a gene encoding a GEF for RhoA. These data support the idea that a systematic search for mutations and/or deletions of GEFs in human cancer is promising.

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The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointments due to inadequate ES cell sources.

Left hemisphere stroke frequently leads to limb apraxia, a disorder that has been reported to impact independence in daily life and rehabilitation success. Nonetheless, there is a shortcoming in research and availability of applicable trainings. Further, to date, anosognosia for limb apraxia has largely been neglected. Therefore, we developed a Naturalistic Action Therapy that trains object selection and application with an errorless learning approach and which includes supported self-evaluation. The current study presents the results of two stroke patients participating in the training. The procedure entailed two baseline and one post-training sessions including standardized limb apraxia and anosognosia assessments as well as 18 naturalistic action tasks. The training consisted of 15 sessions during which 4-6 of the 18 naturalistic action tasks (e.g., pour water into a glass, make a phone call) were trained. Both patients showed improvement in trained and untrained tasks as well as in standardized apraxia and anosognosia assessments. Training effects appeared strongest for the trained items. The procedure is documented in detail and easy to administer and thus may have the potential to be applied by relatives. The results of this pilot-study are promising and suggest that the approach is suitable for further evaluation.

The applicability of T cell receptor (TCR) D␦2D␦3 junctionalThe usefulness of the D␦2D␦3 marker for MRD study has not regions for the detection of minimal residual disease (MRD) been adequately addressed as yet. was examined in childhood acute lymphoblastic leukemiaWe report here on the application of D␦2D␦3 junctional was identified in 29 T-ALL cases. Three patients exhibited D␦2D␦3 recombinations in both alleles. Sequence analysis of D␦2D␦3 junctions revealed extensive diversity due to the random insertion and deletion of nucleotides at the joining site. Materials and methodsPCR analysis utilizing allele-specific probes or oligonucleotides generated on the basis of D␦2D␦3 junctional sequences reached a sufficient sensitivity of 10 −4 to 10 −5 in the majority of viduals. Methods and criteria for the definition ofKeywords: TCR ␦ gene; D␦2D␦3 rearrangement; minimal residual immunophenotypes have been previously described. 10,11 Leudisease; acute lymphoblastic leukemia kemia cell samples contained more than 90% of blasts. Introduction Southern blot analysisThe majority of acute lymphoblastic leukemia (ALL) patients are High molecular weight genomic DNA was prepared from characterized by a unique pattern of immunoglobulin (Ig) and/or cryopreserved BM and PB cells. Ten micrograms of DNA were T cell receptor (TCR) gene recombinations. 1,2 Various PCR stradigested with BglII or HindIII, separated on 0.6% agarose gel tegies utilizing V(D)J junctions of rearranged Ig or TCR loci as and transferred on to nylon membranes (Nytran 13N; clonospecific markers have been introduced for the detection Schleicher & Schuell, Dassel, Germany). After hybridization of minimal residual disease (MRD) in ALL. Current PCR protousing the TCRDJ1 probe 1,7 (kindly provided by Prof JJM van cols allow the reliable detection of one neoplastic cell among Dongen, Erasmus University Rotterdam, The Netherlands) the 10 4 to 10 6 normal cells and are expected to provide novel crifilters were washed and exposed on X-ray film as previously teria for the individualization of treatment modalities. 3,4 described. 12 V␦2D␦3 as well as D␦2D␦3 recombinations constitute preferential TCR␦ gene rearrangements in ALL, namely in precursor-B ALL. [5][6][7] Both their frequency in precursor-B ALL and Sequence analysis of D␦2D␦3 junctions extensive junctional diversity due to the deletion and random insertion of nucleotides at the joining sites favor the usage of these rearrangements as tumor-specific markers for the detecSequences of the primers used for amplification or sequence analysis of D␦2D␦3 junctions have been tion of MRD. 8,9 Previous MRD studies focused on the applications of V␦2D␦3 rearrangements as allele-specific probes.described elsewhere, 12-14 except for the D␦1-5′ primer (5′-ACTCCATGTTCAAATAGATATAGTATT-3′). To isolate D␦2D␦3 junctions for consecutive sequence analyses we used primer D␦1-5′ and the biotinylated D␦3-3′ primer. PCR, isolation of biotinylated amplification products and consecutiveCorrespondence: T Seriu Received 13 January 1997; ac...