Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones.

BACKGROUND: Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. RESULTS: Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. CONCLUSIONS: Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus.

Genomic structure, mapping, activity and expression of fibroblast growth factor 17.

Fibroblast growth factors are essential molecules for development. Here we characterize Fgfl7, a new member of the fibroblast growth factor (FGF) family. The Fgfl7 gene maps to mouse chromosome 14 and is highly conserved between mouse and human (93% identity). It exhibits 60% amino acid identity with Fgf8 and 50% identity with Fgf8. Both Fgf8 and Fgf17 have a similar structure and a similar pattern of alternative splicing in the 5' coding region. When expressed in 3T3 fibroblasts, mouse FGF17 is transforming, indicating that it can activate the 'c' splice form of either FGF receptor (FGFR) one or two. During midgestation embryogenesis, in situ hybridization analysis localized Fgf17 expression to specific sites in the midline structures of the forebrain, the midbrain-hindbrain junction, the developing skeleton and in developing arteries. Comparison to Fgf8 revealed a striking similarity in expression patterns, especially in the central nervous system (CNS), suggesting that both genes may be important for CNS development, although Fgf17 is expressed somewhat later than Fgf8. In the developing skeleton, both genes are expressed in costal cartilage while Fgf8 is preferentially expressed in long bones. In the developing great vessels Fgfl7 is preferentially expressed, suggesting that it may have a more prominent role in vascular growth.

Fibroblast growth factor-8 expression is regulated by intronic engrailed and Pbx1-binding sites.

Fibroblast growth factor-8 (FGF8) plays a critical role in vertebrate development and is expressed normally in temporally and spatially restricted regions of the vertebrate embryo. We now report on the identification of regions of Fgf8 important for its transcriptional regulation in murine ES cell-derived embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5' region of Fgf8 was shown to contain promoter activity. A 0.8-kilobase fragment derived from the large intron of Fgf8 was found to enhance human growth hormone expressed from the Fgf8 promoter 3-4-fold in an orientation dependent manner. The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and Engrailed transcription factors. Gel shift and Western blot experiments documented the presence of these transcription factors in nuclear extracts from ES cell embryoid bodies. In vitro mutagenesis of the Engrailed or Pbx1 site demonstrated that these sites modulate the activity of the intronic fragment. In addition, in vitro mutagenesis of both Engrailed and Pbx1 sites indicated that other unidentified sites are responsible for the transcriptional enhancement observed with the intronic fragment.

MMTV-Fgf8 transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia.

Prior studies have identified Fibroblast Growth Factor-8 (Fgf8) as a possible proto-oncogene in mouse mammary tumorigenesis. We now report on the generation of two types of Fgf8 transgenic mice that each utilize the mouse mammary tumor virus (MMTV) promoter. The first transgene (MMTV-Fgf8b) results in the overexpression of the FGF8b isoform exclusively. Male and female MMTV-Fgf8b transgenic mice are viable and fertile. RNA for FGF8b is detected in mammary gland and salivary gland tissues of transgenic mice by Northern blot analysis. Nearly 85% of breeding transgenic female mice developed mammary lobular adenocarcinomas by 12 months of age, while no tumors developed in non-transgenic littermates. Salivary gland tumors occurred in some animals, always in association with mammary tumors. Several MMTV-Fgf8b transgenic mice had lung metastases at necropsy. The second transgene (MMTV-Fgf8) uses the entire Fgf8 gene and potentially encodes all FGF8 isoforms. Fgf8 is expressed by this transgene in several tissues in addition to those described above, notably the ovaries. The two MMTV-Fgf8 founders developed mammary ductal adenocarcinomas at five and eight months of age, and both displayed ovarian stromal hyperplasia. The founders expressing either transgene did not successfully nurse their pups. These results demonstrate that production of FGF8b, and possibly other FGF8 isoforms, in the mammary and salivary glands contributes to oncogenesis, and that ovarian expression results in stromal hyperplasia.

T gamma/delta hepatosplenic lymphoma in a heart transplant patient after an Epstein-Barr virus positive lymphoproliferative disorder: a case report.

BACKGROUND: An unusual case of a peripheral T-cell lymphoma of T gamma/delta hepatosplenic type (Tgamma/deltaHSL) that arose in a child 5 years after she received a heart transplant and 9 months after she developed Epstein-Barr virus (EBV) positive, B-cell lymphoid hyperplasia involving the tonsils is presented. The majority of the reported cases of Tgamma/deltaHSL have been described in young adult men without antecedent immunodeficiency; several well documented cases of Tgamma/deltaHSL in the posttransplant setting have been described previously, but none has been described in a child (or an adult) with a previously diagnosed EBV+ B-cell lymphoid hyperplasia. METHODS: Standard histologic, immunohistochemical, flow cytometric, and molecular genetic techniques were used in the evaluation of diagnostic material. RESULTS: The patient's Tgamma/deltaHSL involved the spleen in a predominantly cordal pattern, and infiltrated the liver in an exclusively sinusoidal distribution. Bone marrow involvement was focal and interstitial. In all locations, malignant cells were of intermediate or large size and had oval nuclei with coarse chromatin, with a scant or moderate amount of eosinophilic cytoplasm. This Tgamma/deltaHSL expressed the characteristic CD2+, CD3+, [CD4- CD8-], Tdelta1+ phenotype, and malignant cells also expressed the natural killer cell marker CD56. Cytogenetic studies demonstrated isochromosome 7q with the addition of trisomy 8 as the tumor progressed. Southern blot analysis demonstrated clonal rearrangements of the gamma, delta, and beta loci of the T-cell receptor but did not identify EBV DNA within the tumor cells. CONCLUSIONS: This case highlights the fact that a full range of lymphoid proliferations is possible in the posttransplantation period, and that a prior diagnosis of a B-cell disorder does not preclude the development of a subsequent T-cell posttransplant lymphoproliferative disorder (PTLD), which should be formally evaluated, especially if clinical circumstances appear atypical for a PTLD of the "usual" (EBV-related, B-cell) type.

Pathogenesis of ectrodactyly in the Dactylaplasia mouse: aberrant cell death of the apical ectodermal ridge.

Dactylaplasia, or Dac, was recently mapped to the distal portion of mouse chromosome 19 and shown to be inherited as an autosomal semi-dominant trait characterized by missing central digital rays. The most common locus for human split hand split foot malformation, also typically characterized by missing central digital rays, is 10q25, a region of synteny to the Dac locus. The Dac mouse appears to be an ideal genotypic and phenotypic model for this human malformation syndrome. Several genes lie in this region of synteny, however, only Fibroblast Growth Factor 8, or Fgf-8, has been implicated to have a role in limb development. We demonstrate that the developmental mechanism underlying loss of central rays in Dac limbs is dramatic cell death of the apical ectodermal ridge, or AER. This cell death pattern is apparent in E10.5-11.5 Dac limb buds stained with the supravital dye Nile Blue Sulfate. We demonstrate that Fgf8 expression in wild type limbs colocalizes spatially and temporally with AER cell death in Dac limbs. Furthermore, in our mapping panel, there is an absence of recombinants between Fgf-8 and the Dac locus in 133 backcross progeny with a median linkage estimate of approximately 0.5 cM. Thus, our results demonstrate that cell death of the AER in Dac limbs silences the role of the AER as key regulator of limb outgrowth, and that Fgf-8 is a strong candidate for the cause of the Dac phenotype.

Npm3: a novel, widely expressed gene encoding a protein related to the molecular chaperones nucleoplasmin and nucleophosmin.

We report the cloning and initial characterization of the cDNAs, gene, and pseudogene of Npm3, a novel murine gene that encodes a protein related to the nuclear chaperone phosphoproteins, nucleoplasmin and nucleophosmin. Npm3 is located approximately 5 kb upstream of Fgf8 on mouse Chromosome 19 and consists of six exons spanning 2 kb. The first five exons code for an acidic protein of 19.0 kDa that contains a potential nuclear localization signal and potential phosphorylation sites for several kinases. Npm3 was expressed in all mouse tissues examined. On the basis of the similarity of Npm3 to nucleoplasmin and nucleophosmin in amino acid sequence, protein features, and exon structure, we propose that Npm3 is a new member of, and may share basic functions with, the nucleoplasmin/ nucleophosmin family of molecular chaperone proteins.

Overlapping expression and redundant activation of mesenchymal fibroblast growth factor (FGF) receptors by alternatively spliced FGF-8 ligands.

FGF-8 is a member of the family of fibroblast growth factors and is expressed during vertebrate embryo development. Eight potential FGF-8 isoforms are generated by alternative splicing in mice, several of which are expressed during embryogenesis in epithelial locations. The significance of the multiple isoforms is currently unknown. In this report, we investigate the expression patterns and the specificity of the FGF-8 isoforms for known fibroblast growth factor (FGF) receptors. RNAs for seven of the eight potential isoforms are present at multiple sites of embryonic Fgf8 expression. None of the FGF-8 isoforms exhibited activity when assayed with BaF3 cells expressing the "b" splice forms of FGF receptors 1-3, which are mostly expressed in epithelial tissues. Mesenchymally expressed "c" splice forms of FGF receptors 2 and 3 and FGF receptor 4 were activated by several FGF-8 isoforms. These findings are consistent with the hypothesis that the multiple FGF-8 isoforms are functionally redundant and function to signal in paracrine (epithelial to mesenchymal) contexts.

Using technology to enhance the writing processes of students with learning disabilities.

This article reviews the ways that computers can support writing by students with learning disabilities, with an emphasis applications that go beyond word processing. Following an overview of research on word processing is a discussion of software assists with the basic processes of transcription and sentence generation, including spelling checkers, speech synthesis, prediction, and grammar and style checkers. Next, applications that support the cognitive processes of planning are review including prompting programs, outlining and semantic mapping software, and multimedia applications. Finally, the use of computer networks to support collaboration and communication with diverse audiences is addressed.

Structure and sequence of human FGF8.

Recent evidence indicates that Fgf8 is expressed during vertebrate development in multiple locations involved in the patterning and outgrowth of important embryo structures. Cloning and analysis of the murine gene revealed at least eight potential protein isoforms that share a common carboxyl region, encoded by exons 2 and 3, but possess different amino termini, generated by alternative splicing of RNA encoded by multiple 5' exons (exons 1A, 1B, 1C, and 1D). We now report the cloning and sequence of the human FGF8 gene. Human FGF-8 isoforms are identical to their murine counterparts in the common carboxyl region. Four of the human isoforms are identical to, or very similar to, the murine isoforms in the amino termini. However, four of the potential murine isoforms do not have corresponding human isoforms due to marked sequence divergence, leading to a blocked reading frame in exon 1B of FGF8. The lack of the four murine isoforms in humans raises the question of their function in murine development.

Receptor specificity of the fibroblast growth factor family.

Fibroblast growth factors (FGFs) are essential molecules for mammalian development. The nine known FGF ligands and the four signaling FGF receptors (and their alternatively spliced variants) are expressed in specific spatial and temporal patterns. The activity of this signaling pathway is regulated by ligand binding specificity, heparan sulfate proteoglycans, and the differential signaling capacity of individual FGF receptors. To determine potentially relevant ligand-receptor pairs we have engineered mitogenically responsive cell lines expressing the major splice variants of all the known FGF receptors. We have assayed the mitogenic activity of the nine known FGF ligands on these cell lines. These studies demonstrate that FGF 1 is the only FGF that can activate all FGF receptor splice variants. Using FGF 1 as an internal standard we have determined the relative activity of all the other members of the FGF family. These data should serve as a biochemical foundation for determining developmental, physiological, and pathophysiological processes that involve FGF signaling pathways.

Roles for FGF8 in the induction, initiation, and maintenance of chick limb development.

We provide evidence that FGF8 serves as an endogenous inducer of chick limb formation and that its expression in the intermediate mesoderm at the appropriate time and place to trigger forelimb development is directly linked to the mechanism of embryonic kidney differentiation. One function of the limb inducer is to initiate Fgf8 gene expression in the ectoderm overlying the prospective limb-forming territories. FGF8 secreted by the ectoderm then appears to initiate limb bud formation by promoting outgrowth of and Sonic hedgehog expression in the underlying lateral plate mesoderm. FGF8 also maintains mesoderm outgrowth and Sonic hedgehog expression in the established limb bud. Our data thus point to FGF8 as a key regulator of limb development that not only induces and initiates the formation of a limb bud, but also sustains its subsequent development.

The importance of phase I/II trials in pediatric oncology.

Clinical trials in pediatric oncology over the past 30 years have led to the situation today where most children with newly diagnosed cancer can be treated effectively, and many are cured. Despite this dramatic improvement in outcome for many children diagnosed with cancer, about 30-40% of children will die of their disease [1]. Although some attempts have been made to improve outcome by increasing the dose intensity of existing therapies, intolerable side effects and marginal increases in cancer cell kill limit this approach. Clearly, effective new anti-cancer agents are necessary to significantly improve the survival and quality of life in children with cancer. Well-organized pediatric Phase I trials to establish the maximum tolerated dose (MTD), and Phase II trials to establish efficacy, are critical to the identification of new anti-cancer agents.

Assignment of FGF8 to human chromosome 10q25-q26: mutations in FGF8 may be responsible for some types of acrocephalosyndactyly linked to this region.

Emerging evidence suggests that Fgf8, a recently identified member of the fibroblast growth factor family, plays an important role in outgrowth and patterning of the face, limbs, and central nervous system of the vertebrate embryo. We report the mapping of FGF8 to human chromosome 10q25-q26, using Southern blot analyses of genomic DNAs from rodent/human somatic cell hybrid lines. Apert, Crouzon, Jackson-Weiss, and Pfeiffer syndromes are craniosynostoses genetically linked in part to 10q25-q26 and are associated with point mutations in the extracellular domain of FGFR2. Given the assignment to the same chromosomeal band(s) as FGFR2 and the probable ligand-receptor relationship of the gene products of FGF8 and FGFR2, we hypothesize that some cases of these craniosynostoses linked to 10q25-q26 that do not have mutations in FGFR2 may involve mutations in FGF8.

FGF-8 isoforms activate receptor splice forms that are expressed in mesenchymal regions of mouse development.

The Fgf8 gene is expressed in developing limb and craniofacial structures, regions known to be important for growth and patterning of the mouse embryo. Although Fgf8 is alternatively spliced to generate at least 7 secreted isoforms that differ only at their mature amino terminus, the biological significance of these multiple isoforms is not known. In this report, we demonstrate that multiple FGF-8 isoforms are present at sites of Fgf8 expression during mouse development. To address the possibility that the FGF-8 isoforms might interact with different fibroblast growth factor receptors, we prepared recombinant FGF-8 protein isoforms. We examined the ability of these proteins to activate alternatively spliced forms of fibroblast growth factor receptors 1-3, and fibroblast growth factor receptor 4. Recombinant FGF-8b and FGF-8c activate the 'c' splice form of FGFR3, and FGFR4, while FGF-8b also efficiently activates 'c' splice form of FGFR2. No activity could be detected for recombinant or cell expressed FGF-8a. Furthermore, none of the isoforms tested interact efficiently with 'b' splice forms of FGFR1-3, or the 'c' splice form of FGFR1. These results indicate that the FGF-8b and FGF-8c isoforms, produced by ectodermally derived epithelial cells, interact with mesenchymally expressed fibroblast growth factor receptors. FGF-8b and FGF-8c may therefore provide a mitogenic signal to the underlying mesenchyme during limb and craniofacial development.

Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro.

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.

FGF-8 isoforms differ in NIH3T3 cell transforming potential.

We previously identified Fgf-8 as a frequently activated gene in tumors from mouse mammary tumor virus-infected Wnt-1 transgenic mice, suggesting that Fgf-8 is a proto-oncogene. We further determined that multiple, secreted protein isoforms that differ at their mature amino termini are encoded by alternatively spliced mRNAs transcribed from the gene. We now present evidence that there are differences in the potency of NIH3T3 cell transformation displayed by three of the FGF (fibroblast growth factor)-8 isoforms. We find that stable transfection of a cDNA for the FGF-8b isoform leads to marked morphological transformation of NIH3T3 cells and rapid tumorigenicity of the transfected cells in nude mice. In contrast, transfection of a cDNA for the FGF-8a or FGF-8c isoform results in moderate morphological changes in the NIH3T3 cells, and the transfected cells are weakly tumorigenic in nude mice. All three transfections result in cells that express comparable amounts of Fgf-8 mRNA and that produce the FGF-8 protein isoforms. The morphological changes observed in NIH3T3 cells can be reproduced by the addition of recombinant FGF-8 protein isoforms to the culture medium. Therefore, these results indicate that there are differences in the potency of transformation of NIH3T3 cells by FGF-8 protein isoforms and suggest that these FGF-8 isoforms may have different in vivo functions.

Fgf-8, activated by proviral insertion, cooperates with the Wnt-1 transgene in murine mammary tumorigenesis.

We have used mouse mammary tumor virus (MMTV) infection of Wnt-1 transgenic mice to accelerate mammary tumorigenesis and to molecularly tag insertionally activated proto-oncogenes that cooperate oncogenically with Wnt-1 (G. M. Shackleford, C. A. MacArthur, H. C. Kwan, and H. E. Varmus, Proc. Natl. Acad. Sci. USA 90:740-744, 1993). Here we report the identification and characterization of a 31-kb genomic locus that contains clonal MMTV integrations in 8 of 80 mammary tumors from MMTV-infected Wnt-1 transgenic mice. Two genes were identified within this locus, one of which was transcriptionally activated by MMTV insertions. This activated gene is identical to androgen-induced growth factor (AIGF/Fgf-8) (A. Tanaka, K. Miyamoto, N. Minamino, M. Takeda, B. Sato, H. Matsuo, and K. Matsumoto, Proc. Natl. Acad. Sci. USA 89:8928-8932, 1992), the eighth member of the fibroblast growth factor (FGF) family. Transcriptional activation of Fgf-8 was found in all tumors with MMTV insertions in this locus. Fgf-8 mRNA was absent in normal mammary glands and was detected only in adult testis and ovary and in midgestational embryos. The sequences of Fgf-8 genomic and cDNA clones revealed five coding exons, in contrast to the three coding exons found in other FGF genes. cDNAs encoding three isoforms of the FGF-8 protein were isolated. The three corresponding mRNAs resulted from the alternative use of two 5' splice sites and two 3' splice sites for the second and third exons, respectively. These results implicate Fgf-8 as the third FGF gene found to cooperate with Wnt-1 in MMTV-induced murine mammary tumorigenesis, suggesting that FGFs and Wnts are strong collaborators in this process.

Student Assistant for Learning from Text (SALT): a hypermedia reading aid.

Student Assistant for Learning from Text (SALT) is a software system for developing hypermedia versions of textbooks designed to help students with learning disabilities and other low-achieving students to compensate for their reading difficulties. In the present study, 10 students with learning disabilities (3 young women and 7 young men ages 15 to 17) in Grades 9 and 10 read passages from a science textbook using a basic computer version and an enhanced computer version. The basic version included the components found in the printed textbook (text, graphics, outline, and questions) and a notebook. The enhanced version added speech synthesis, an on-line glossary, links between questions and text, highlighting of main ideas, and supplementary explanations that summarized important ideas. Students received significantly higher comprehension scores using the enhanced version. Furthermore, students preferred the enhanced version and thought it helped them learn the material better.