EphA receptors and ephrin-A ligands exhibit highly regulated spatial and temporal expression patterns in the developing olfactory system

The spatiotemporal expression patterns of the chemorepulsive EphA receptors, EphA4 and EphA7, and three ephrins-A2, A4 and A5, were examined in the developing rat primary olfactory system. Unlike the visual system that has simple and stable gradients of Ephs and ephrins, the olfactory system demonstrates complex spatiotemporal expression patterns of these molecules. Using immunohistochemistry, we demonstrate that expression of these molecules is dynamic and tightly regulated both within and between different cell types. We reveal restricted targeting of these proteins within subcellular compartments of some neurons. EphA4, ephrin-A2 and ephrin-A5 were expressed by primary olfactory axons during the embryonic formation of the olfactory nerve. There were no gradients in expression along the rostrocaudal or ventrodorsal axes in the nasal cavity and olfactory bulb. However, during the early neonatal period, axons expressing different levels of ephrin-A5 sorted out and terminated in a subpopulation of glomeruli that were mosaically dispersed throughout the bulb. The expression of EphA4 and ephrin-A2 was dramatically down-regulated on all axons during the early neonatal period of glomerular formation. The uniform co-expression of receptors and ligands before glomerular formation suggests they play a generic role in axon-axon interactions in the olfactory nerve and nerve fibre layer. In contrast, loss of EphA4 from axons during glomerular formation may facilitate the interaction of ephrin-A5 with Eph receptors on target cells in the bulb. While EphA4, EphA5 and EphA7 are not mosaically expressed by bulbar neurons, other Eph receptors may have expression patterns complementary to the ephrin-A5-positive subpopulation of glomeruli. (C) 2002 Elsevier Science B.V. All rights reserved.

Concordance of iron indices in homozygote and heterozygote sibling pairs in hemochromatosis families: implications for family screening

Background/Aims: Concordance of iron indices between same sex siblings homozygous for the cysteine-to-tyrosine substitution at amino acid 282 (C282Y) mutation suggests that the variable phenotype in hereditary hemochromatosis is caused by genetic factors. Concordance of iron indices between same-sex heterozygous sibling pairs would provide further evidence of genetic modifiers of disease expression, and guidance for family screening strategies of subjects heterozygous for the C282Y mutation. Methods: We compared the iron indices of 35 C282Y homozygous and 35 C282Y heterozygous same-sex sibling pairs. To clarify whether concordance between siblings was due to environmental or genetic factors we compared the iron indices of 164 C282Y homozygous-normal, same-sex dizygotic twins. Results: Serum ferritin (r = 0.50, P = 0.003), hepatic iron concentration (r = 0.61, P = 0.025) and hepatic iron index (r = 0.67, P = 0.01) were highly concordant in C282Y homozygotes. Heterozygote siblings were concordant for serum ferritin (r = 0.76, P = 0.0001) and transferrin saturation (r = 0.79, P = 0.0001). Homozygote-normal same-sex dizygotic twins were concordant for serum ferritin (r = 0.62, P = 0.0001) but not for transferrin saturation. Conclusions: Concordance of iron indices exists in C282Y homozygote and heterozygote sibling pairs. Siblings of expressing C282Y heterozygotes require phenotypic assessment. These data provide evidence for modifying genes influencing disease expression in hemochromatosis. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Ephrin-A5 induces rounding, blebbing and deadhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling

Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions, membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.

A prostaglandin F2a Analog induces suppressors of the cytokine signalling-3 expression n the corpus luteum of the pregnant rat: A potential new mechanism in luteolysis

PRL and placental lactogen (PL) play key roles in maintaining the rodent corpus luteum through pregnancy. Suppressors of cytokine signaling (SOCS) have been shown to decrease cell sensitivity to cytokines, including PRL, and so here we have addressed the issue of whether luteolysis induced by prostaglandin F-2alpha (PGF(2alpha)) might up-regulate SOCS proteins to inhibit PRL signaling. In d 19 pregnant rats, cloprostenol, a PGF(2alpha) analog, rapidly induced transcripts for SOCS-3 and, to a lesser extent, SOCS-1. We also found increased SOCS-3 protein in the ovary by immunoblot and in the corpus luteum by immunohistochemistry. Increased SOCS-3 expression was preceded by an increase in STAT3 tyrosine phosphorylation 10 min after cloprostenol injection and was maintained for 4 h, as determined by gel shift and immunohistochemistry. Induction of SOCS-3 was accompanied by a sharp decrease in active STAT5, as determined by gel-shift assay and by loss of nuclear localized STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to down-regulation of PRL receptor. Therefore, induction of SOCS-3 by PGF(2alpha) may be an important element in the initiation of luteolysis via rapid suppression of luteotropic support from PL.

Temporal and spatial expression of fibroblast growth factor receptor 4 isoforms in murine tissues

Fibroblast growth factor receptors (FGFRs) undergo highly regulated spatial and temporal changes of expression during development. This study describes the use of quantitative reverse transcriptase-polymerase chain reaction and immunochemistry to assess the changes in expression of FGFR4 as compared to its FGFR4-17a and -17b isoforms in mouse tissues, from early embryogenesis through to adulthood. Compared to FGFR4, the expression of the isoforms is more restricted at all developmental stages tested. The reverse transcriptase-polymerase chain reaction demonstrated that FGFR4 is expressed in more tissue types than either of its isoforms: it was found predominantly in lung, liver, brain, skeletal muscle and kidney, whereas the FGFR4-17a form was detected in lung and skeletal muscle, and the FGFR4-17b form only in lung, liver, skeletal muscle and kidney. Immunohistochemistry confirmed strong FGFR4-17b expression in the postnatal lung. When combined, the results suggest that FGFR4 variants play important roles particularly in lung and skeletal muscle development.

Complementary and layered expression of Ephs and Ephrins in developing mouse inner ear

The distributions of the Eph-class receptors EphA4 and EphB 1, and their ligands ephrin-A2, ephrin-B1, and ephrin-B2, were analysed by immunostaining in the mouse inner ear. Complementary patterns of EphA4 and its potential ligand ephrin-A2 were found, with ephrin-A2 in many of the structures lining the cochlear duct and within the cochlear nerve cells, and EphA4 in the deeper structures underlying the cochlear duct and in the cells lining the nerve pathway. EphB1 and its potential ligands ephrin-B1 and ephrin-B2 showed a segregated layered expression in the lateral wall of the cochlear duct (the external sulcus), which together with EphA4 expressed in the area, form a four-layered structure with an alternating pattern of receptors and ligands in the different layers. This arrangement gives the potential for different bidirectional Eph-mediated interactions between each of the layers. The results suggest that the Eph system in the cochlea may have a role in maintaining cell segregation during phases of cochlear development. (C) 2002 Wiley-Liss, Inc.

Prolonged retention after aggregation into secretory granules of human R183H-growth hormone (GH), a mutant that causes autosomal dominant GH deficiency type II

Human R183H-GH causes autosomal dominant GH deficiency type II. Because we show here that the mutant hormone is fully bioactive, we have sought to locate an impairment in its progress through the secretory pathway as assessed by pulse chase experiments. Newly synthesized wild-type and R183H-GH were stable when expressed transiently in AtT20 cells, and both formed equivalent amounts of Lubrol-insoluble aggregates within 40 min after synthesis. There was no evidence for intermolecular disulfide bond formation in aggregates of wild-type hormone or the R183H mutant. Both wildtype and R183H-GH were packaged into secretory granules, assessed by the ability of 1 mm BaCl2 to stimulate release and by immunocytochemistry. The mutant differed from wildtype hormone in its retention in the cells after packaging into secretory granules; 50% more R183H-GH than wild-type aggregates were retained in AtT20 cells 120 min after synthesis, and stimulated release of R183H-GH or a mixture of R183H-GH and wild-type that had been retained in the cell was reduced. The longer retention of R183H-GH aggregates indicates that a single point mutation in a protein contained in secretory granules affects the rate of secretory granule release.

Decreased expression of the Id3 gene at 1p36.1 in ovarian adenocarcinomas

The molecular events that drive the initiation and progression of ovarian adenocarcinoma are not well defined. We have investigated changes in gene expression in ovarian cancer cell lines compared to an immortalized human ovarian surface epithelial cell line (HOSE) using a cDNA array. We identified 17 genes that were under-expressed and 10 genes that were over-expressed in the cell lines compared to the HOSE cells. One of the genes under-expressed in the ovarian cancer cell lines, Id3, a transcriptional inactivator, was selected for further investigation. Id3 mRNA was expressed at reduced levels in 6 out of 9 ovarian cancer cell lines compared to the HOSE cells while at the protein level, all 7 ovarian cancer cell lines examined expressed the Id3 protein at greatly reduced levels. Expression of Id3 mRNA was also examined in primary ovarian tumours and was found in only 12/38 (32%) cases. A search was conducted far mutations of Id3 in primary ovarian cancers using single stranded conformation polymorphism (SSCP) analysis. Only one nucleotide substitution, present also in the corresponding constitutional DNA, was found in 94 ovarian tumours. Furthermore no association was found between LOH at 1p36 and lack of expression of Id3. These data suggest that Id3 is not the target of LOH at 1p36. (C) 2001 Cancer Research Campaign.

Facilitation of renal autoregulation by angiotensin II is mediated through modulation of nitric oxide

Aims: This study was designed to investigate the influence of angiotensin II (Ang II) and nitric oxide (NO) on autoregulation of renal perfusion. Methods: Autoregulation was investigated in isolated perfused kidneys (IPRK) from Sprague-Dawley rats during stepped increases in perfusion pressure. Results: Ang II (75-200 pM) produced dose-dependent enhancement of autoregulation whereas phenylephrine produced no enhancement and impaired autoregulation of GFR. Enhancement by Ang II was inhibited by the AT(1) antagonist, Losartan, and the superoxide scavenger, Tempol. Under control conditions nitric oxide synthase (NOS) inhibition by 10 muM N-omega-nitro-L-arginine methyl ester (L-NAME) facilitated autoregulation in the presence of non-specific cyclooxygenase (COX) inhibition by 10 muM indomethacin. Both COX and combined NOS/COX inhibition reduced the autoregulatory threshold concentration of Ang II. Facilitation by 100 pM Ang II was inhibited by 100 muM frusemide. Methacholine (50 nM) antagonised Ang II-facilitated autoregulation in the presence and absence of NOS/COX inhibition. Infusion of the NO donor, 1 muM sodium nitroprusside, inhibited L-NAME enhancement of autoregulation under control conditions and during Ang II infusion. Conclusions: The results suggest than an excess of NO impairs autoregulation under control conditions in the IPRK and that endogenous and exogenous NO, vasodilatory prostaglandins and endothelium-derived hyperpolarizing factor (EDHF) activity antagonise Ang II-facilitated autoregulation. Ang II also produced a counterregulatory vasodilatory response that included prostaglandin and NO release. We suggest that Ang II facilitates autoregulation by a tubuloglomerular feedback-dependent mechanism through AT(1) receptor-mediated depletion of nitric oxide, probably by stimulating generation of superoxide.

Gene Expression in Brain: A Window on Ethanol Dependence, Neuroadaptation, and Preference

This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarrays, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl.

Site-directed mutagenesis of the ATM promoter: Consequences for response to proliferation and ionizing radiation

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. (C) 2003 Wiley-Liss, Inc.

Expression of ephs and ephrins in developing mouse inner ear

Levels of expression of mRNAs encoding the different Ephs and ephrins were measured by semi-quantitative reverse-transcription polymerase chain reaction in developing mouse whole inner ears, and in dissected fractions of the neonatal mouse inner ear. Nineteen of the 24 known Ephs and ephrins were surveyed. The results showed that between embryonic age (E) 11.5 days and E12.5, levels increased 10-300 times per unit of tissue. In neonatal mice, the fraction containing combined organ of Corti and spiral ganglion showed relatively strong expression of EphA4, EphB3, ephrin-A3, ephrin-B2 and ephrin-B3. In the lateral wall, EphA4, ephrin-A3 and ephrin-B2 were strongly expressed, while ephrin-A3 was particularly strongly expressed in utricular and saccular sensory epithelia. The results suggest that the Ephs and ephrins are likely to play a part in the differentiation of the structures of the inner ear, and show which Ephs and ephrins are most likely to play important roles in the different structures. (C) 2003 Elsevier Science B.V. All rights reserved.

Chiral resolution of hexaamine cobalt(III) cages: Substituent effects on chiral discrimination

Chiral resolution of the cobalt cage complexes [Co(diNOsar)](3+) and [Co(diAMsarH(2))](5+) have been achieved by selective crystallization with the anion bis-mu-(R),(R)-tartratodiantimonate(III) ([Sb-2(R,R-tart)(2)](2-)) and also by column chromatography with Na-2[Sb-2(R, R-tart)(2)] as eluent. The X-ray crystal structures of Lambda-[ Co(diNOsar)][Sb-2(R, R-tart)(2)] Cl . 7H(2)O and Delta-[Co(diAMsarH(2))][Sb-2(R, R-tart)(2)](2)Cl . 14H(2)O are reported, which reveal an unexpected reversal of chiral discrimination when the cage substituent is changed from nitro (Lambda-enantiomer) to ammonio (Delta-enantiomer) and shows that the ammonio- substituted cage is capable of forming a three-point hydrogen-bonding interaction with each complex anion, whereas the nitro analogue can only form two hydrogen bonds with each [Sb-2(R, R-tart)(2)](2-) anion. During cation exchange chromatography of the racemic cobalt cage complexes with Na-2[Sb-2(R, R-tart)(2)] as eluent, Lambda-[Co(diNOsar)](3+) elutes first, which implies a tighter ion pairing interaction than for the Delta-enantiomer. On the other hand, Delta-[Co(diAMsarH(2))](5+) elutes first during chromatography under identical conditions, which is also consistent with a preferred outer-sphere complex formed between Delta-[Co(diAMsarH(2))](5+) and [Sb-2(R, R-tart)(2)](2-) relative to Lambda-[Co(diAMsarH(2))](5+) and [Sb-2(R,R-tart)(2)](2-).