Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis, We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display, We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development, This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats, Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo, As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis. (C) 1997 Wiley-Liss, Inc.

HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Immunity to asexual blood stage malaria and vaccine approaches

The development of a malaria vaccine seems to be a definite possibility despite the fact that even individuals with a life time of endemic exposure do not develop sterile immunity. An effective malaria vaccine would be invaluable in preventing malaria-associated deaths in endemic areas, especially amongst children less than 5 years of age and pregnant women. This review discusses our current understanding of immunity against the asexual blood stage of malaria - the stage that is responsible for the symptoms of the disease - and approaches to the design of an asexual blood stage vaccine.

Identification of regions within the third FnIII-like domain of the IL-5R alpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5R alpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5R alpha (hIL-5R alpha) and mouse IL-5R alpha (mIL-5R alpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5R alpha but not the mIL-5R alpha. By using chimeras of the mIL-5R alpha and hIL-5R alpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5R alpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5R alpha to mIL-5R alpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity, These results show that regions of the third FnIII domain of IL-5R alpha are involved in binding, in addition to the regions in domains one and two of the IL-5R alpha that were identified in an earlier study. (C) 2000 Academic Press.

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). I, contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-KB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

Identification and analysis of chromodomain-containing proteins encoded in the mouse transcriptome

The chromodomain is 40-50 amino acids in length and is conserved in a wide range of chromatic and regulatory proteins involved in chromatin remodeling. Chromodomain-containing proteins can be classified into families based on their broader characteristics, in particular the presence of other types of domains, and which correlate with different subclasses of the chromodomains themselves. Hidden Markov model (HMM)-generated profiles of different subclasses of chromodomains were used here to identify sequences encoding chromodomain-containing proteins in the mouse transcriptome and genome. A total of 36 different loci encoding proteins containing chromodomains, including 17 novel loci, were identified. Six of these loci (including three apparent pseudogenes, a novel HP1 ortholog, and two novel Msl-3 transcription factor-like proteins) are not present in the human genome, whereas the human genome contains four loci (two CDY orthologs and two apparent CDY pseuclogenes) that are not present in mouse. A number of these loci exhibit alternative splicing to produce different isoforms, including 43 novel variants, some of which lack the chromodomain. The likely functions of these proteins are discussed in relation to the known functions of other chromodomain-containing proteins within the same family.

Gene expression during early ascidian metamorphosis requires signaling by Hemps, an EGF-like protein

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.

The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF)

A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric alpha-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and H-1-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblasts. As we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.

Structural analysis of three His32 mutants of DsbA: Support for an electrostatic role of His32 in DsbA stability

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys3O-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of similar to 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 Variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys3O. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.

MUC13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells

Transmembrane mucins are glycoproteins involved in barrier function in epithelial tissues. To identify novel transmembrane mucin genes, we performed a tblastn search of the GenBank(TM) EST data bases with a serine/ threonine-rich search string, and a rodent gene expressed in bone marrow was identified. We determined the cDNA sequence of the human orthologue of this gene, MUC13, which localizes to chromosome band 3q13.3 and generates 3.2-kilobase pair transcripts encoding a 512-amino acid protein comprised of an N-terminal mucin repeat domain, three epidermal growth factor-like sequences, a SEA module, a transmembrane domain, and a cytoplasmic tail (GenBank(TM) accession no. AF286113), MUC13 mRNA is expressed most highly in the large intestine and trachea, and at moderate levels in the kidney, small intestine, appendix, and stomach, In situ hybridization in murine tissues revealed expression in intestinal epithelial and lymphoid cells. Immunohistochemistry demonstrated the human MUC13 protein on the apical membrane of both columnar and goblet cells in the gastrointestinal tract, as well as within goblet cell thecae, indicative of secretion in addition to presence on the cell surface. MUC13 is cleaved, and the beta -subunit containing the cytoplasmic tail undergoes homodimerization, Including MUC13, there are at least five cell surface mucins expressed in the gastrointestinal tract.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Protein disulfide isomerase: the structure of oxidative folding

Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.

Growth hormone, insulin-like growth factor I and cell proliferation in the mouse blastocyst

The role of growth hormone (GH) in embryonic growth is controversial, yet preimplantation embryos express GH, insulin-like growth factor I (IGF-I) and their receptors. In this study, addition of bovine GH doubled the proportion of two-cell embryos forming blastocysts and increased by about 25% the number of cells in those blastocysts with a concentration-response curve showing maximal activity at 1 pg bovine GH ml(-1), with decreasing activity at higher and lower concentrations. GH increased the number of cells in the trophectoderm by 25%, but did not affect the inner cell mass of blastocysts. Inhibition of cell proliferation by anti-GH antiserum indicated that GH is a potent autocrine or paracrine regulator of the number of trophectoderm cells in vivo. Type 1 IGF receptors (IGF1R) were localized to cytoplasmic vesicles and plasma membrane in the apical domains of uncompacted and compacted eight-cell embryos, but were predominantly apparent in cytoplasmic vesicles of the trophectoderm cells of the blastocyst, similar to GH receptors. Studies using alphaIR3 antiserum which blocks ligand activation of IGF1R, showed that IGF1R participate in the autocrine or paracrine regulation of the number of cells in the inner cell mass by an endogenous IGF-I-IGF1R pathway. However, alphaIR3 did not affect GH stimulation of the number of trophectoderm cells. Therefore, CH does not use secondary actions via embryonic IGF-I to modify the number of blastocyst cells. This result indicates that GH and IGF-I act independently. GH may selectively regulate the number of trophectoderm cells and thus implantation and placental growth. Embryonic GH may act in concert with IGF-I, which stimulates proliferation in the inner cell mass, to optimize blastocyst development.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFSP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-5 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGK at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P