A novel nonsense mutation in the EpCAM gene in a patient with congenital tufting enteropathy.

OBJECTIVES: Tufting enteropathy (TE) is a classical congenital disorder of the intestinal mucosa causing protracted diarrhea in infancy as a result of a dysfunctional epithelial cell barrier, which is mainly caused by mutations in the EpCAM gene and expression of a nonfunctional epithelial cell adhesion molecule in the intestine. We report here a novel nonsense mutation in a patient suspected of having TE, resulting in a complete absence of EpCAM in duodenal enterocytes. METHODS: A patient presenting with congenital diarrhea and suspected of having TE was screened for EpCAM mutations, and duodenal biopsies were stained for EpCAM using immunohistochemistry analysis. RESULTS: We identified a novel homozygous nonsense mutation in the EpCAM gene in a patient suspected of having TE, causing a complete loss of EpCAM expression in duodenal enterocytes. CONCLUSIONS: With screening analysis for EpCAM mutations and immunohistochemistry for EpCAM expression in duodenal enterocytes, we found a novel homozygous mutation in a patient with classical protracted diarrhea in infancy finally diagnosed as TE, which results in a complete absence of EpCAM and in dysfunctional barrier formation in duodenal enterocytes.

DEF-3(g16/NY-LU-12), an RNA binding protein from the 3p21.3 homozygous deletion region in SCLC.

DEF-3(g16/NY-LU-12) encodes a novel RNA binding protein isolated by positional cloning from an SCLC homozygous deletion region in 3p21.3 and, in parallel, as a differentially expressed gene during myelopoiesis from FDCPmix-A4 cells. DEF-3(g16/NY-LU-12) is ubiquitously expressed during mouse embryogenesis and in adult organs while human hematopoietic tissues showed differential expression. The mouse and human proteins are highly conserved containing two RNA recognition motifs (RRMs) and other domains associated with RNA binding and protein-protein interactions. A database search identified related proteins in human, rat, C. elegans and S. pombe including the 3p21.3 co-deleted gene, LUCA15. Recombinant proteins containing the RRMs of DEF-3(g16/NY-LU-12) and LUCA15 specifically bound poly(G) RNA homopolymers in vitro. These RRMs also show similarity to those of the Hu protein family. Since anti-Hu RRM domain antibodies are associated with an anti-tumor effect and paraneoplastic encephalomyelitis, we tested sera from Hu syndrome patients with the RRMs of DEF-3(g16/NY-LU-12) and LUCA15. These were non-reactive. Thus, DEF-3(g16/NY-LU-12) and LUCA15 represent members of a novel family of RNA binding proteins with similar expression patterns and in vitro RNA binding characteristics. They are co-deleted in some lung cancers and immunologically distinct from the Hu proteins.

Endothelial monocyte-activating polypeptide-II (EMAP-II): a novel inducer of lymphocyte apoptosis.

The novel, proinflammatory cytokine endothelial monocyte-activating polypeptide-II (EMAP-II) was first found in tumor cell supernatants. EMAP-II is closely related or identical to the p43 auxiliary protein of the multisynthase complex, which is involved in protein synthesis. In vitro, EMAP-II induces procoagulant activity, increased expression of E- and P-selectins and tumor necrosis factor receptor-1, and ultimately, programmed cell death (apoptosis) in cultured endothelial cells. EMAP-II is also chemotactic for monocytes and neutrophils. However, the role of the p43/EMAP-II cytokine form in tumors is not understood. We hypothesized an immune-regulatory role within neoplastic tissues and investigated its effects on lymphocytes. EMAP-II causes a dose-dependent inhibition of proliferation and apoptosis in Jurkat T cells and mitogen-activated peripheral blood mononuclear cells. Coculture with DLD-1 colorectal cancer cells or media conditioned by these cells induces apoptosis in Jurkat cells, which is partially reversed by antibodies against EMAP-II. Our data suggest that EMAP-II constitutes a component of a novel, immunosuppressive pathway in solid tumors, which is not normally expressed outside the cell but in tumors, may be subject to abnormal processing and released from tumor cells.

Lateral self-assembly of E-cadherin directed by cooperative calcium binding.

We report the Ca2+ binding characteristics of recombinant Ecad12, a construct spanning the first two repeats of epithelial cadherin, and demonstrate the links between Ca2+ binding and dimer formation. Sedimentation equilibrium and dynamic light scattering experiments show that weak dimerization of Ecad12 occurs in the presence of 10 mM Ca2+ (KdP = 0.17 mM), while no appreciable dimer formation was detected in the absence of Ca2+. Ca2+-induced dimerization was also observed in electron microscopy images of Ecad12. We conclude from Ca2+ titration experiments monitored by tryptophan fluorescence and flow dialysis that dimerization does not affect the equilibrium binding constant for Ca2+. However, the value of the Hill coefficient for Ca2+ binding increases from 1.5 to 2.4 as the protein concentration increases, showing that dimer formation largely contributes to the cooperativity in Ca2+ binding. Based on these observations and previous crystallographic studies, we propose that calcium acts more likely as a geometrical aligner ensuring the proper assembly of cadherin molecules, rather than a simple adhesive.

Low energy loss electron microscopy of chromophores.

A novel prism-mirror-prism imaging electron spectrometer with 1 eV energy resolution for a transmission electron microscope permits imaging with spectral energies corresponding to light-optical colour absorptions. The instrument selects the molecular orbital excitations of natural chromophores or of specific dyes normally used in biological light microscopy for delineation and chemical identification, but images them with electron microscopic detail. Heavy atom contrast agents customarily used in electron microscopy are not required. The first results exploit the intrinsic red colour of hematin molecules to demonstrate the potential of the technique and address its spatial resolution. Glycosaminoglycans in cartilage stained with Alcian blue are selectively depicted in situ by means of the electron-induced molecular absorption of this chromophore. Thus, with the use of specific colours the direct or indirect analysis of local chemistry by electron microscopy is possible, and can be carried out with a depiction of spatial detail as small as 16 A, or at least 100-fold finer than observed by light microscopy.

Imaging of calcium in biological specimens: role of electron energy loss spectroscopy.

The regulation of intracytoplasmic calcium is an essential mechanism involved in many normal cytological functions. Disorders of calcium regulation are coupled to numerous pathological conditions. Calcium is such a universal participant that knowledge of its fine movements and sites of action is essential for the understanding of many biological phenomena at the cellular level. Techniques for the measurement of intracellular calcium have been dramatically improved, and we have now achieved great sensitivity to concentration changes as well as a very high time resolution. Recent advances in calcium research technology have concentrated on imaging methods permitting the visualization of calcium movements and localization of its cytoplasmic distribution. At the light microscopical level, it is now possible to trace the fluctuations of intracellular free calcium in a living cell, using sensitive calcium binding fluorescent indicators. Electron microscopic techniques are therefore necessary for the ultrastructural localization of cytoplasmic calcium stores. Electron energy loss spectroscopy is at present the most sensitive microanalytical mode in electron microscopy. It is capable of simultaneously producing high resolution images and elemental information. It therefore has application in calcium research if the specimens are prepared by an appropriate method. This review has two objectives: (1) To place electron energy loss imaging in its proper perspective in calcium research. (2) To suggest that freeze-dried embedded biological materials are appropriate for high resolution energy loss imaging of calcium.

Electron energy loss spectroscopic imaging in biology.

One of the goals in biology is to relate the ultrastructure with the movement of elements to understand better physiological and pathophysiological mechanisms. Electron energy loss spectroscopy (EELS) imaging, which was developed in the last decade, appears to be an ideal technique to make such correlation. EELS takes advantage of the energy distribution of transmitted electrons which interacted with the specimen. All these electrons are collected and can be displayed as an energy loss spectrum for analytical purposes. Images can be produced from selected regions from the energy distribution allowing the mapping of specific elements. The main advantage of EELS imaging in biology is its spatial resolution of 0.5 nm or less and its great sensitivity allowing nearly a single atom detectability. The limitations reside essentially in specimen preparation. In order to obtain optimal results with EELS imaging, only very thin specimens can be used. This restricts the way biological specimens can be prepared. This is a real challenge for the analysis of diffusible elements. Other limitations reside in the difficulty of quantifying the results obtained. This is greatly due to the fact that theoretical considerations still have to be experimentally validated. The purpose of this review is not to repeat in length the principle of EELS but to emphasize its achievement in biology and to assess the present advantages and limitations. Also, as EELS imaging is still in its development phase, results already obtained are a strong indication that this technique has a great prospect in the analysis of dynamic biological processes.

Freestanding lipid bilayers as substrates for electron cryomicroscopy of integral membrane proteins.

Phospholipid bilayers, 40 A thick, were generated as electron microscope substrates by submerging copper grids overlaid with holey plastic through a lipid monolayer on a water surface. Previously formed proteoliposomes containing single-particle membrane proteins in their bilayers were then fused into the newly formed bilayer substrate. To demonstrate this methodology, multi-drug resistance protein P-glycoprotein was incorporated into these bilayers and imaged by fixed beam microscopy and scanning transmission electron microscopy.

Experimental ionization cross-sections of phosphorus and calcium by electron spectroscopic imaging.

The absolute partial electron scattering cross-section for the phosphorus L2,3-shell ionization was measured by electron spectroscopic imaging using poliovirus as a primary standard. The equivalent calcium cross-section was obtained in relation to phosphorus using the stoichiometric ratio for these two elements in hydroxyapatite, Ca10(PO4)6(OH)2. At 80 keV, the partial cross-section of phosphorus was 2.26 x 10(-20) and 2.68 x 10(-20) cm2/atom for poliovirus and hydroxyapatite, respectively, at 150 eV loss for a 15-eV energy window and an acceptance angle of 15 mrad. Under the same conditions the calcium cross-section was 0.49 x 10(-20) cm2/atom at 360 eV loss. The experimental values are slightly higher than the theoretical cross-sections calculated either by hydrogenic or Hartree-Slater approaches.

Towards practical soft X-ray spectromicroscopy of biomaterials.

Scanning transmission X-ray microscopy (STXM) is being developed as a new tool to study the surface chemical morphology and biointeractions of candidate biomaterials with emphasis on blood compatible polymers. STXM is a synchrotron based technique which provides quantitative chemical mapping at a spatial resolution of 50 nm. Chemical speciation is provided by the near edge X-ray absorption spectral (NEXAFS) signal. We show that STXM can detect proteins on soft X-ray transparent polymer thin films with monolayer sensitivity. Of great significance is the fact that measurements can be made in situ, i.e. in the presence of an overlayer of the protein solution. The strengths, limitations and future potential of STXM for studies of biomaterials are discussed.

Localization of chromophore absorption signals in TEM with an improved prism-mirror-prism filter.

A corrected prism-mirror-prism electron energy filter with curved entrance and exit faces was designed and incorporated into a Zeiss EM902 transmission electron microscope. The installation of the new filter required little modification to the existing microscope geometry and electronics. The filter had an energy resolution of 1.1 eV over the full image plane (acceptance half angle 10 mradian). The improved energy resolution was applied in studies of the low electron energy loss region that includes molecular orbital excitations or chromophore energy absorptions. Chromophore signal behaviour under electron irradiation was characterized using embedded crystals of hematin and of the dye mercury orange. Images of these crystals confirmed the expected decrease in signal intensity on shifting the selected energy loss from the region of molecular orbital excitations (less than approximately 5 eV) to higher energy losses. Electron irradiation-induced fading of the chromophore signal from hematin and mercury orange yielded similar 1/e dose values of 1.1 x 10(5) e(-) nm(-2) and 1.4 x 10(5) e(-) nm(-2) respectively. In a cellular context, chromophore signals were obtained from energy-filtered images of RIF-1 cell sections containing the photosensitizer chlorin e6 and from sections of BS-C-1 cells with cytoskeletal labelling via FITC-conjugated antibodies. The respective signals were extracted using a dose-dependent method or a shift in selected energy. Chromophore signal distributions were in agreement with fluorescence light microscopic images, but provided detail at higher spatial resolution.

Filaments of surfactant protein A specifically interact with corrugated surfaces of phospholipid membranes.

Pulmonary surfactant, a mixture of lipids and surfactant proteins (SPs), plays an important role in respiration and gas exchange. SP-A, the major SP, exists as an octadecamer that can self-associate to form elongated protein filaments in vitro. We have studied here the association of purified bovine SP-A with lipid vesicle bilayers in vitro with negative staining with uranyl acetate and transmission electron microscopy. Native bovine surfactant was also examined by transmission electron microscopy of thinly sectioned embedded material. Lipid vesicles made from dipalmitoylphosphatidylcholine and egg phosphatidylcholine (1:1 wt/wt) generally showed a smooth surface morphology, but some large vesicles showed a corrugated one. On the smooth-surfaced vesicles, SP-As primarily interacted in the form of separate octadecamers or as multidirectional protein networks. On the surfaces of the striated vesicles, SP-As primarily formed regularly spaced unidirectional filaments. The mean spacing between adjacent striations and between adjacent filaments was 49 nm. The striated surfaces were not essential for the formation of filaments but appeared to stabilize them. In native surfactant preparations, SP-A was detected in the dense layers. This latter arrangement of the lipid bilayer-associated SP-As supported the potential relevance of the in vitro structures to the in vivo situation.

A novel murine P-450 gene, Cyp4a14, is part of a cluster of Cyp4a and Cyp4b, but not of CYP4F, genes in mouse and humans.

Genomic clones for Cyp4a12 and a novel member of the murine Cyp4a gene family were isolated. The novel gene, designated Cyp4a14, has a GC rich sequence immediately 5' of the transcription start site, and is similar to the rat CYP4A2 and CYP4A3 genes. The Cyp4a14 gene spans approximately 13 kb, and contains 12 exons; sequence similarity to the rat CYP4A2 gene sequence falls off 300 bp upstream from the start site. In view of the known sex-specific expression of the rat CYP4A2 gene, the expression and inducibility of Cyp4a14 was examined. The gene was highly inducible in the liver when mice were treated with the peroxisome proliferator, methylclofenapate; induction levels were low in control animals and no sex differences in expression were observed. By contrast, the Cyp4a12 RNA was highly expressed in liver and kidney of control male mice but was expressed at very low levels in liver and kidney of female mice. Testosterone treatment increased the level of this RNA in female liver slightly, and to a greater extent in the kidney of female mice. In agreement with studies on the cognate RNA, expression of Cyp4a12 protein was male-specific in the liver of control mice and extremely high inducibility of Cyp4a10 protein, with no sex differences, was also demonstrated. In view of the overlapping patterns of inducibility of the three Cyp4a genes, we investigated whether the three genes were co-localized in the genome. Two overlapping yeast artificial chromosome (YAC) clones were isolated, and the three Cyp4a genes were shown to be present on a single YAC of 220 kb. The Cyp4a genes are adjacent to the Cyp4b1 gene, with Cyp4a12 most distant from Cyp4b1. The clustering of these two gene subfamilies in the mouse was replicated in the human, where the CYPA411 and CYP4B1 genes were present in a single YAC clone of 440 kb. However, the human CYP4F2 gene was mapped to chromosome 19. Phylogenetic analysis of the CYP4 gene families demonstrated that CYP4A and CYP4B are more closely related than CYP4F.