Continuous high-repetition-rate operation of collisional soft-x-ray lasers with solid targets.

We have generated a laser average output power of 2 microW at a wavelength of 13.9 nm by operating a tabletop laser-pumped Ni-like Ag laser at a 5 Hz repetition rate, using a solid helicoidal target that is continuously rotated and advanced to renew the target surface between shots. More than 2 x 10(4) soft-x-ray laser shots were obtained by using a single target. Similar results were obtained at 13.2 nm in Ni-like Cd with a Cd-coated target. This scheme will allow uninterrupted operation of laser-pumped tabletop collisional soft-x-ray lasers at a repetition rate of 10 Hz for a period of hours, enabling the generation of continuous high average soft-x-ray powers for applications.

A crucial function of PDGF in TGF-beta-mediated cancer progression of hepatocytes.

Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-beta. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-beta-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-beta, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-beta superfamily of cytokines, TGF-beta1, -beta2 and -beta3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-alpha receptor revealed decreased TGF-beta-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-beta-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.

Human RERE is localized to nuclear promyelocytic leukemia oncogenic domains and enhances apoptosis.

RE repeats encoded (RERE) was identified recently as a protein with high homology to the atrophin-1 protein, which appears to be causal in the hereditary neurodegenerative disorder termed dentatorubral-pallidoluysian atrophy (DRPLA) caused by an abnormal glutamine expansion. We have independently identified RERE in a search for genes localized to the translocation breakpoint region at chromosome 1p36.2 in the neuroblastoma cell line NGP. Here we show that neuroblastoma tumor cell lines display reduced abundance of RERE transcripts. Furthermore, we detected RERE protein mainly in the nucleus, where it colocalizes with the promyelocytic leukemia protein in promyelocytic leukemia oncogenic domains (PODs). Overexpression of RERE recruits a fraction of the proapoptotic protein BAX to PODS: This observation correlates with RERE-induced apoptosis, which occurs in a caspase-dependent manner. These results identify RERE as a novel component of PODs and suggest an important role of RERE in the control of cell survival.

Independent regulation of the two Pax5 alleles during B-cell development.

The developmental control genes of the Pax family are frequently associated with mouse mutants and human disease syndromes. The function of these transcription factors is sensitive to gene dosage, as mutation of one allele or a modest increase in gene number results in phenotypic abnormalities. Pax5 has an important role in B-cell and midbrain development. By following the expression of individual Pax5 alleles at the single-cell level, we demonstrate here that Pax5 is subject to allele-specific regulation during B-lymphopoiesis. Pax5 is predominantly transcribed from only one allele in early progenitors and mature B cells, whereas it switches to a biallelic transcription mode in immature B cells. The allele-specific regulation of Pax5 is stochastic, reversible, independent of parental origin and correlates with synchronous replication, in contrast with imprinted and other monoallelically expressed genes. As a consequence, B-lymphoid tissues are mosaics with respect to the transcribed Pax5 allele, and thus mutation of one allele in heterozygous mice results in deletion of the cell population expressing the mutant allele due to loss of Pax5 function at the single-cell level. Similar allele-specific regulation may be a common mechanism causing the haploinsufficiency and frequent association of other Pax genes with human disease.

Molecular cloning and expression analysis of five novel genes in chromosome 1p36.

The human chromosome 1p36 region displays frequent nonrandom chromosomal deletions and translocations in a number of human malignancies; these are thought to inactivate tumor suppressor genes. To identify these putative tumor suppressors we employed exon trapping, cDNA selection, and zoo blot analysis to clone five new genes located in 1p36. Two of these represent novel genes and were designated C1orf1 and xylan 1,4-beta-xylosidase 1 (XBX1). Two further genes represented new members of known gene families: PTPRZ2 was a tyrosine phosphatase and FRAP2 represented a FKBP12-rapamycin-associated protein. The fifth gene identified, ENO1L1, was significantly homologous to c-myc promoter binding protein, MBP-1, and to enolase 1 (ENO1). It colocalized with alpha enolase (ENO1) on a single P1 clone. ENO1L1 differed from both ENO1 and MBP-1 in the organization of its 5' untranslated sequences. Second, MBP-1 contained two single-base insertions not present in either ENO1 or ENO1L1 sequences, which led to a shift in the MBP-1 reading frame. Expression analysis revealed two brain-specific transcripts of 7.9 and 6.5 kb for PTPRZ2. In contrast, C1orf1, FRAP2, ENO1L1, and XBX1 appeared to be expressed ubiquitously in the tissues tested, with transcript sizes of 4.5, 8.7, 1.75, and 4.5 kb, respectively. Using fluorescence in situ hybridization, we mapped the five novel genes relative to chromosome 1p36 breakpoints present in three established tumor cell lines and one nontumor cell line. The karyotypic abnormalities in these cell lines were exploited as chromosomal landmarks; we could thus show that the telomere to centromere gene order was PTPRZ2-(MBP-1/ENO1/ENO1L1)-(C1orf1/XBX1)-+ ++FRAP2. The localization of these genes to a chromosomal region that is prone to deletions in human cancers makes them potential candidate tumor suppressors.

Cloning and characterization of CpG islands of the human chromosome 1p36 region.

CpG islands were identified and localized to chromosome 1p36 by means of pulsed-field gel blot hybridization with 1p36-specific microclone probes. Five CpG islands, designated CpG17, CpG28, CpG60, CpG112a, and CpG112b, were molecularly cloned from corresponding cosmids. All five islands are associated with transcribed sequences, as shown by RNA blot hybridizations. Screening of cDNA libraries with the island-specific genomic probes led to the isolation of two cDNA clones to date. These encode the human transcription factor E2F-2 and the dominant-negative helix-loop-helix gene ID3, respectively. Pulsed-field gel electrophoresis analysis also revealed that these two genes are located next to each other at a distance of about 25 kb.

Analysis of location and integrity of the human PITSLRE (p58(cdc2L1)) genes in neuroblastoma cell genomes.

The family of PITSLRE kinase genes, located in chromosome 1p36, has recently been associated with neuroblastoma tumorigenesis. In order to evaluate the role of these genes as putative tumor suppressor genes, we have analyzed the integrity of the coding region in primary tumors and its location relative to a neuroblastoma consensus deletion. A subset of aggressive neuroblastoma tumors with allelic loss of different parts of chromosome 1p were investigated. Single-strand conformation polymorphism (SSCP), heteroduplex (HD) and sequencing analysis of tumor DNA did not reveal any significant changes in the coding region. In particular, a primary tumor with an interstitial allelic deletion in 1p36 did not reveal concomitant loss of heterozygosity of the PITSLRE gene region when analyzed with a C/T DNA sequence polymorphism in exon 5 of PITSLRE1. FISH analysis on neuroblastoma cell lines with small interstitial deletions and with a balanced translocation in 1p36 revealed that the PITSLRE gene cluster was localized distal to the neuroblastoma consensus deletion. against an involvement of the PITSLRE genes in neuroblastoma tumorigenesis.

Determination and regional assignment of grouped sets of microclones in chromosome 1pter-p35.

In an approach to mapping physically the most distal 30 Mb of human chromosome 1p, region-specific clone libraries were generated by microdissection and microcloning. PFGE blot hybridization of single or low-copy microclones against rare-cutter digests of genomic DNA revealed physical linkage for groups of markers. Supplementary PFGE analysis of 31 1p36-p35-specific probes for genetically mapped loci established a total of 15 grouped sets, consisting of altogether 69 markers. Twelve of the grouped sets were located in 1pter-p36.12, as revealed by microcell hybrid mapping; the remaining three were localized proximal to 1p36.12. Regional assignment and ordering of most grouped sets was achieved either by evaluating the included genetic markers or by fluorescence in situ hybridization of representative probes. The genomic extent of individual grouped sets encompassed between 1100 and 2100 kb, covering a total of approximately 22 Mb of the distal chromosome 1p region. One particular grouped set was shown to contain seven polymorphic marker loci that were previously suggested to be distributed across the entire 1pter-p35 region. The increase in the number of hybridization marker probes in 1p36 and their physical mapping is expected to facilitate positional cloning experiments in this region; in particular, the construction of clone contigs may be greatly facilitated.

Conservation of a maternal-specific methylation signal at the human IGF2R locus.

The human IGF2R gene has been reported to be either biallelically or very rarely monoallelically expressed, in contrast to the maternally expressed mouse counterpart. We describe here an analysis of the 5' portion of the human IGF2R gene and show that it contains a maternally methylated CpG island in the second intron. A similar maternally methylated intronic element has been proposed to be the imprinting box for the mouse gene and although the relevance of this element has yet to be directly demonstrated, methylation has been reported to be essential to maintain allele-specific expression of imprinted genes. Allelic expression analysis of human IGF2R in 70 lymphoblastoid cell lines identified only one line showing monoallelic expression. Thus, in this tissue monoparental methylation of the IGF2R gene does not correlate with allele-specific expression. We also confirm here that the human IGF2R gene is located in an asynchronously replicating chromosomal region, as are all other imprinted genes so far analyzed. The mouse and human IGF2R intronic CpG islands both contain numerous large direct repeats that are methylated following maternal, but not paternal, transmittance. Thus features that attract maternal-specific methylation are conserved between the mouse and human genes. Since these intronic CpG islands share organizational rather than sequence homology, this suggests that secondary DNA structure may play a role in attracting a maternal methylation imprint.

Expression of the helix-loop-helix gene Id3 during murine embryonic development.

The family of dominant-negative helix-loop-helix (dnHLH) transcriptional modulators consists of four mammalian genes known to date: Id1, Id2, Id3 (Id3 was formerly designated HLH462 or HEIR1 or HLH1R21), and Id4. We have performed a detailed study of Id3 expression in mouse embryos in order to gain insight into the possible developmental control functions of this gene and to compare its expression to that of other mammalian dnHLH genes. Employing RNA in situ hybridization on sectioned mouse embryos of gestational stages E9.5-17.5 and neonatal head, we generally detected a high abundance of transcripts at early stages that gradually declined at most sites toward E15.5. Expression of Id3 in embryos was detected in brain, spinal cord, olfactory system, branchial arches, limbs, sclerotome, endocardiac cushions, the outer lining of the gut, lung, retina, the collecting system of the kidney, and in tooth anlagen. Although the abundance of mRNA decreased toward later stages in most tissues, it remained high in teeth and kidney. This expression pattern suggests that Id3 functions both in undifferentiated tissues and in organs which are in the process of differentiation. When compared to the expression of other dnHLH genes, it becomes obvious that the pattern of Id3 mainly coincides with that of Id1. This may reflect a partial redundancy in gene function. Furthermore, previous experiments suggested a mutually exclusive expression of the proto-oncogene N-myc an Id3. The results of the present study confirm these data.

The gene coding for the B cell surface protein CD19 is localized on human chromosome 16p11.

The CD19 gene codes for one of the earliest markers of the human B cell lineage and is a target for the B lymphoid-specific transcription factor BSAP (Pax-5). The transmembrane protein CD19 has been implicated in controlling proliferation of mature B lymphocytes by modulating signal transduction through the antigen receptor. In this study, we have employed Southern blot and fluorescence in situ hybridization analyses to localize the CD19 gene to human chromosome 16p11.

[Reproductive and developmental toxicity studies of lactitol (NS-4) (3)--Teratogenicity study in rabbits by oral administration].

Lactitol, a hepatic encephalopathy drug, was administered by oral gavage to pregnant New Zealand White rabbits during organogenesis from day 6 to day 18 of gestation inclusive, at dosages of 0, 0.25, 0.75 or 4.5 g/kg/day. On day 29 of gestation, females were killed to allow examination of their uterine contents. There was a slight reduction in food intake and faecal output among females receiving 4.5 g/kg. One female receiving 4.5 g/kg aborted following a prolonged period of weight loss. No adverse effects on litter parameters were recorded that could be attributed to treatment. Foetal morphogenesis was unaffected by treatment with lactitol. The results show that no-effect dose levels of lactitol are 0.75 g/kg in mother rabbits for general toxicity and for reproductive functions, and 4.5 g/kg for their fetuses.

Large-scale isolation of human 1p36-specific P1 clones and their use for fluorescence in situ hybridization.

A series of 80 microclone probes derived from the chromosomal region 1p36 was used to isolate corresponding clones from the ICRF human P1 library (see Francis et al., this issue). Hybridization screenings were performed using probe pools on high-density filter grids. A total of 87 P1 clones specific for 1p36 were isolated. This large-scale approach allowed a detailed evaluation of the complexity, quality, and utility of this library. The isolated P1 clones were used both for size determination by pulsed-field gel electrophoresis and as probes for fluorescence in situ hybridization (FISH) analysis. FISH of P1 clones is shown to be both easy and efficient to perform on metaphase chromosomes and interphase nuclei. This observation is expected to reveal new avenues for diagnosis of disease-related chromosomal changes. The use of P1 clones as a tool in clinical and tumor interphase cytogenetics is discussed and compared with FISH data of other long insert clones such as cosmids and YAC clones.

Characterization of a region-specific library of microclones in the vicinity of the Bcg and splotch loci on mouse chromosome 1.

The proximal portion of mouse chromosome 1 harbors a variety of mutant loci that have yet to be characterized at the molecular level. We have constructed a library of genomic DNA fragments from the proximal portion of mouse chromosome 1 by microdissection and microcloning techniques, with the aim of generating genetic markers in close proximity to some of these mutant loci. To facilitate the genetic mapping of 27 microclones from this library, we divided a 56-cM segment of chromosome 1 between the Col3a1 and Ren 1,2 genes into eight intervals defined by anchor loci. Restriction fragment length polymorphisms were determined for each of the microclones and their segregation with the anchor loci was followed in informative animals from a panel of 252 interspecific backcross mice (C57BL/6J x Mus spretus) x C57BL/6J. We were able to assign 26 of 27 (96%) randomly selected microclones to each of the defined chromosome 1 intervals. A total of eight microclones mapped within the large interstitial deletion found in the Spr mouse mutant. Two of these clones were found to be tightly linked to the host resistance locus Bcg and at least one was found to be linked to the neural tube defect mutant splotch. Other clones mapped to intervals containing several other mouse mutants. These novel DNA markers should aid in positional cloning strategies presently employed to identify these mutant loci. These clones should also be useful in the creation of both physical and YAC contiguous maps of the proximal portion of mouse chromosome 1.

A physical map of the region encompassing the human interleukin-1 alpha, interleukin-1 beta, and interleukin-1 receptor antagonist genes.

We have constructed a restriction map of the human genomic region containing the genes encoding the three members of the interleukin-1 (IL-1) family, IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1 ra). For this purpose, pulsed-field gel electrophoresis blots were hybridized with probes derived from the three genes. The genes (IL1A, IL1B, and IL1RN, respectively) were found to map to a common restriction fragment of approximately 430 kb that is flanked by two clusters of sites for methylation-sensitive rare-cutter restriction enzymes (putative CpG islands). A likely third internal CpG island was marked by two rare-cutter sites. CpG and non-CpG-specific enzymes were used to map the three genes. Relative to one terminal CpG island, the three genes were mapped to the following intervals: IL1A was between +0 and +35 kb, IL1B was between +70 and +110 kb, and IL1RN was between +330 and +430 kb.

Chromosomal localization of seven PAX genes and cloning of a novel family member, PAX-9.

In the human paired box-containing (PAX) gene family, only two members, PAX-3 and PAX-6, which are associated with Waardenburg's syndrome and aniridia, respectively have been mapped to human chromosomes. We have now isolated cosmids for six additional human PAX genes (PAX-1,-2,-5,-7,-8,-9) and a polymerase chain reaction fragment for PAX-4. PAX-9 is a novel family member which is closely related in its paired domain to PAX-1. The chromosomal location of all cloned PAX genes was determined by analysis of somatic cell hybrids and (except PAX-4) by fluorescence in situ hybridization to metaphase chromosomes. PAX-1 and PAX-7 map to chromosomal regions containing previously assigned disease loci.