Jacob Nielsen

A Family of Insulin-Like Growth Factor II mRNA-Binding Proteins Represses Translation in Late Development

ABSTRACT Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5′ untranslated regions (5′ UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5′ UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5′ UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 –luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5′ UTR-binding proteins control IGF-II biosynthesis during late mammalian development.

Dwarfism and Impaired Gut Development in Insulin-Like Growth Factor II mRNA-Binding Protein 1-Deficient Mice

ABSTRACT Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). To characterize the physiological role of IMP1, we generated IMP1-deficient mice carrying a gene trap insertion in the Imp1 gene. Imp1−/− mice were on average 40% smaller than wild-type and heterozygous sex-matched littermates. Growth retardation was apparent from E17.5 and remained permanent into adult life. Moreover, Imp1−/− mice exhibited high perinatal mortality, and only 50% were alive 3 days after birth. In contrast to most other organs, intestinal epithelial cells continue to express IMP1 postnatally, and Imp1−/− mice exhibited impaired development of the intestine, with small and misshapen villi and twisted colon crypts. Analysis of target mRNAs and global expression profiling at E12.5 indicated that Igf2 translation was downregulated, whereas the postnatal intestine showed reduced expression of transcripts encoding extracellular matrix components, such as galectin- 1, lumican, tenascin-C, procollagen transcripts, and the Hsp47 procollagen chaperone. Taken together, the results demonstrate that IMP1 is essential for normal growth and development. Moreover, IMP1 may facilitate intestinal morphogenesis via regulation of extracellular matrix formation.

H19 RNA Binds Four Molecules of Insulin-like Growth Factor II mRNA-binding Protein

H19 RNA is a major oncofetal 2.5-kilobase untranslated RNA of unknown function. The maternally expressedH19 gene is located 90 kilobase pairs downstream from the paternally expressed insulin-like growth factor II(IGF-II) gene on human chromosome 11 and mouse chromosome 7; and due to their reciprocal imprinting and identical spatiotemporal expression, it is assumed that the two genes are functionally coupled. Here we show that human H19 RNA contains four attachment sites for the oncofetal IGF-II mRNA-binding protein (IMP) with apparentK d values in the 0.4–1.3 nm range. The multiple attachment sites are clustered within a 700-nucleotide segment encoded by exons 4 and 5. This 3′-terminal segment targets H19 RNA to lamellipodia and perinuclear regions in dispersed fibroblasts where IMP is also localized. The results suggest that IMP participates in H19 RNA localization and provides a link between the IGF-II and H19genes at post-transcriptional events during mammalian development.

A family of IGF-II mRNA binding proteins (IMP) involved in RNA trafficking.

During a search for trans -acting factors associating with insulin-like growth factor II (IGF-II) mRNAs, we recently identified a family of three I GF-II m RNA-binding P roteins (IMP1, IMP2 and IMP3) that exhibit multiple attachments to IGF-II leader 3 mRNA and the reciprocally imprinted H19 RNA. IMPs contain the unique combination of two RNA recognition motifs (RRMs) and four hnRNP K homology (KH) domains. IMP1 is orthologous to the chicken zipcode-binding protein (ZBP-1), and the mouse c-myc coding region determinant-binding protein (CRD-BP) that associates with g -actin and c-myc mRNA, respectively. Moreover, the p62 protein identified in hepatocellular carcinoma represents a splice variant of IMP2, and IMP3 is orthologous to the Xenopus Vegetal 1 RNA-binding protein (Vg1-RBP/Vera). IMPs are produced in a biphasic fashion - initially during the early stages of embryogenesis and subsequently later in development. IMPs and their orthologues are predominantly cytoplasmic and are implicated in the transport of their RNA targets towards the leading edge in somatic cells and to the vegetal pole in Xenopus oocytes, respectively. RNA localization is a conserved mechanism of polarizing genetic information in the establishment of asymmetries during both embryogenesis and adult life, enabling local protein synthesis at final destinations within the cell. The identification of developmentally expressed zipcode-binding proteins indicates that RNA trafficking participates in processes such as cell-growth and migration during embryogenesis.

C-MYC and IGF-II mRNA-binding protein (CRD-BP/IMP-1) in benign and malignant mesenchymal tumors

Mouse coding region determinant–binding (mCRD‐BP) and human IGF‐II mRNA–binding 1 (hIMP‐1) proteins are orthologous mRNA‐binding proteins that recognize c‐myc and IGF‐II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD‐BP/IMP‐1 binds to c‐myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD‐BP/IMP‐1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD‐BP/IMP‐1. In particular, expression was significant in 14 Ewings sarcomas, all of which were positive. The data suggest that hCRD‐BP/IMP‐1 plays a role in abnormal cell proliferation in mesenchymal tumors.

Nuclear transit of human zipcode-binding protein IMP1.

The human IMPs (insulin-like growth factor II mRNA-binding proteins) belong to a vertebrate zipcode-binding protein family consisting of two RNA recognition motifs and four K homology domains and have been implicated in cytoplasmic mRNA localization, turnover and translational control. In the present study, we show that IMP1 is capable of translocating into nuclei of NIH 3T3 fibroblasts and its immunoreactivity is present in the nuclei of human spermatogenic cells. IMP1 does not contain a simple import signal, but nuclear entry was facilitated by disruption of RNA binding and cytoplasmic granule formation. IMP1 contains two NESs (nuclear export signals) within the RNA-binding K homology domains 2 and 4. The former is a leucine-rich leptomycin B-sensitive NES, whereas the latter is a leptomycin B-insensitive NES. Taken together, these results indicate that IMP1 may attach to its target mRNAs in the nucleus and thereby define the cytoplasmic fate of the transcripts.

The biphasic expression of IMP/Vg1-RBP is conserved between vertebrates and Drosophila

The human IGF-II mRNA-binding proteins (IMPs) 1-3, and their Xenopus homologue Vg1 RNA-binding protein (Vg1-RBP) are RNA-binding proteins implicated in mRNA localization and translational control in vertebrate development. We have sequenced the Drosophila homologue (dIMP) of these genes, and examined its expression pattern in Drosophila embryos by in situ hybridization. The study shows that dIMP exhibits a biphasic expression pattern. In the early stages of development, a maternal pool of dIMP mRNA is evenly distributed in the embryo and degraded by the end of stage 4. Expression reappears in the developing central nervous system, where dIMP is expressed throughout neurogenesis. In addition, dIMP is present in the pole cells.

Evidence of orbital B and T cell depletion after rituximab therapy in Graves' ophthalmopathy

lus. No mitoses or necrosis were present. The cytoplasm was eosinophilic and coarsely granular. The granules were periodic acid-Schiff stain-positive, diastase-resistant and stained red–brown with Masson trichrome stain. Immunohistohemically, the tumour cells expressed focal staining for S100 protein and vimentin. Binding of Ki-67 antibodies revealed a low mitotic index (< 1%). The tumour cells were negative in the following immunostains: a-1-antichymotrypsin; CD20; CD31; CD34; CD68; CK7; CKAE-CAM; EMA (epithelial membrane antigen); GCDFP (gross cystic disease fluid protein); inhibin-a; Leu7; MU213-UC; NSE (neuron specific enolase), and SMA (smooth muscle actin). Small nerve branches were observed adjacent to the tumour cells. The section margins showed tumour involvement. Electron microscopy of tumour tissue retrieved from the paraffin block showed numerous secondary lysosomes with undistinguishable content (Fig. 1D) and a small amount of endoplasmatic reticulum. The findings are consistent with a diagnosis of granular cell tumour (GCT). No recurrence was observed in 4 years of follow-up. Granular cell tumour has been reported in almost every part of the body, but is rare in the orbit and ocular adnexae (Ordonez & Mackay 1999); as far as we know, it has never been reported in the lacrimal gland. The term describes a slowgrowing tumour that occurs in patients of all ages, but most commonly during the fourth to sixth decades and twice as often in females as in males (Ordonez & Mackay 1999). In the orbital and periocular locations, the tumour often presents with displacement of the eyeball and ⁄or impairment of eye movements (Jaeger et al. 1987). Several differential diagnostic considerations should be taken into account because some may have clinical implications. Acinic cell carcinoma and oncocytoma may resemble GCT, but the former stains positively for cytokeratin and the granules in the latter represent mitochondria rather than lysosomes (Ellis & Auclair 1995). The treatment of choice is complete surgical removal. Granular cell tumour rarely recurs and the prognosis is good (Ordonez & Mackay 1999). The predominant theory of origin is derivation from Schwann cells. That GCT has been found in almost every tissue and organ confirms that the cell of origin is found everywhere (Ordonez & Mackay 1999). The tumour cells are often located adjacent to small nerve branches (Ordonez & Mackay 1999), as seen in our case. This is consistent with a Schwann cell origin and is further supported by the result of immunohistochemical studies (Le et al. 2004) demonstrating S100 positivity, as in the present case. The GCT granules represent secondary lysosomes and have occasionally been found to contain digested myelin (Jaeger et al. 1987). However, granular cell change can occur in a wide variety of neoplastic conditions and some authors have suggested that the lysosomes may represent a metabolic alteration that is not exclusively associated with Schwann cell tumours (Rosai 1996).

Evaluation of three transportable multigas anesthetic monitors: The Bruel & Kjaer Anesthetic Gas Monitor 1304, the Datex Capnomac Ultima, and the Nellcor N-2500

We compared the performance of three newly developed anesthetic agent (AA) monitors: the Bruel & Kjaer Anesthetic Agent Monitor 1304 (BK 1304), the Datex Capnomac Ultima (ULTIMA), and the Nellcor N-2500 (N-2500). The following were investigated: the linearity and accuracy in measuring AAs, oxygen, carbon dioxide, and nitrous oxide; the linearity and accuracy during warm-up time; the effect of increasing respiratory rate on the accuracy; the consequences of a difference between monitored and delivered AA and of delivering a mixture of AAs; and, finally, the effect of water vapor and alcohol. For all three monitors we found that the accuracy in determining the respiratory and anesthetic gases was sufficient for clinical use (the N-2500 does not measure oxygen). Because of the calibration mixture supplied with the device, however, the ULTIMA recorded values that were 10 to 12% (relative) less than the AA that was present. The BK 1304 had greater accuracy at higher respiratory rates than did the other two monitors, probably favoring its use in pediatric anesthesia. The N-2500 will detect which agent (isoflurane, enflurane, or halothane) is being used, alone or in a mixture. With the two other monitors the user must define which agent is given. In some situations a difference between this and the one actually delivered can theoretically lead to an overdose of AA, with the ULTIMA up to a 14.9 minimal alveolar concentration (MAC) overdose. No interference from alcohol or water vapor in the expired air was found. When the units millimeters of mercury (mm Hg) and kilopascal (kPa) were chosen, the ULTIMA displayed the values in standard temperature pressure dry (STPD) instead of body temperature pressure saturated (BTPS) conditions. Following power-up, some time lapsed before the monitors accurately displayed all variables, shortest with the BK 1304 and ULTIMA (15 min) and longest with the N-2500 (40 min).

Methods to produce calibration mixtures for anesthetic gas monitors and how to perform volumetric calculations on anesthetic gases

A simple procedure for making calibration mixtures of oxygen and the anesthetic gases isoflurane, enflurane, and halothane is described. One to ten grams of the anesthetic substance is evaporated in a closed, 11,361-cc glass bottle filled with oxygen gas at atmospheric pressure. The carefully mixed gas is used to calibrate anesthetic gas monitors. By comparison of calculated and measured volumetric results it is shown that at atmospheric conditions the volumetric behavior of anesthetic gas mixtures can be described with reasonable accuracy using the ideal gas law. A procedure is described for calculating the deviation from ideal gas behavior in cases in which this is needed.