Eugene Daniels

Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells

Biliary glycoprotein (Bgp) is a member of the immunoglobulin superfamily and the carcinoembryonic antigen family. Previous studies have shown that Bgp functions as an intercellular adhesion molecule and a canalicular bile salt transporter. Moreover, we and others demonstrated that Bgp can inhibit colonic and prostatic tumor cell growth in vivo, through a mechanism which depends on sequences present in its cytoplasmic domain. In this study, we have examined the possibility that the cytoplasmic domain of Bgp can interact with signal transduction molecules. We showed that tyrosine phosphorylated Bgp, expressed in mouse colon carcinoma CT51 cells, could reversibly associate with protein tyrosine phosphatase SHP-1. Mutation of either of two tyrosine residues present in the cytoplasmic domain of Bgp abrogated SHP-1 binding, suggesting that this association was mediated by both tyrosine residues. Similarly, we noted that either of the two SH2 domains of SHP-1 could bind tyrosine phosphorylated Bgp in vitro. It is therefore conceivable that some of the functions of Bgp are mediated through its ability to induce intracellular protein tyrosine dephosphorylation.

Progranulin (acrogranin/PC cell-derived growth factor/granulin-epithelin precursor) is expressed in the placenta, epidermis, microvasculature, and brain during murine development.

The growth factor progranulin (acrogranin/PC‐derived growth factor/granulin‐epithelin precursor) promotes onset of blastocyst cavitation and is required for neonatal hypothalamic sexual differentiation. Little is known, however, of the range of developmental processes in which it is involved. We used in situ hybridization to investigate progranulin expression in murine embryos. Progranulin mRNA is expressed in maternal and embryonic components during early establishment of pregnancy. Abundant expression is observed in the early decidualizing uterine stroma and glands. In the embryo, the trophoblast giant cells at the interface of placental exchange sites (both choriovitelline and chorioallantoic placenta) show strong expression. The gastrulating epiblast and mesenchyme (intraembryonic and extraembryonic mesenchyme) all revealed activity. The allantois and yolk sac mesenchyme (site of early hemopoiesis) were positive, as were later phases of active vessel formation (pia mater of brain, epicardium of the heart). In the urogenital system, it was expressed in Sertoli cells and in kidney tubules. It was highly expressed in proliferating epidermal cells. During epidermal appendage formation, the early epithelial bud was positive, but the forming duct and differentiating adjacent mesenchyme was negative. It is widely distributed during central nervous system development and the peripheral nervous system (dorsal root ganglia and sympathetic ganglia). Based on the pattern of progranulin gene expression, we propose proliferative and developmental roles for progranulin in establishing pregnancy, during gastrulation, and during embryonic development of the epidermis, nervous system, blood vessel, formation, and spermatogenesis. Developmental Dynamics 593–599, 2003.

Essential function of PTP-PEST during mouse embryonic vascularization, mesenchyme formation, neurogenesis and early liver development

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST(-/-) embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5-10.5 PTP-PEST(-/-) embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.

Expression of the Wdr9 gene and protein products during mouse development

Human WDR9 has been mapped to chromosome 21, within one of the Down syndrome (DS) critical regions. Here, we study the expression pattern of the murine Wdr9 gene and its protein product. We show that Wdr9 is broadly expressed in the mouse embryo by means of in situ hybridization and immunohistochemistry. Wdr9 expression levels are dynamic during embryonic development as revealed by Northern blot analysis. We further show that WDR9 is a nuclear protein associated with BRG1, a SWI/SNF complex component. We also demonstrate that a polyglutamine‐containing region of the protein functions as a transcriptional activation domain. We propose that WDR9 is a transcriptional regulator involved in chromatin remodeling through the action of two bromodomains and contacts to the SWI/SNF complex. These results may provide a molecular basis for the association of WDR9 with DS. Developmental Dynamics 227:608–614, 2003.

Biliary glycoprotein 1 expression during embryogenesis: correlation with events of epithelial differentiation, mesenchymal-epithelial interactions, absorption, and myogenesis.

Biliary glycoprotein (Bgp1), a carcinoembryonic antigen‐related family member of the immunoglobulin superfamily, is involved in normal and neoplastic events. Analysis of Bgp1 expression throughout post‐implantation mouse embryogenesis using reverse transcription‐polymerase chain reactions, immunostaining with anti‐Bgp1 monoclonal antibodies, and in situ hybridization with specific Bgp1 cDNA fragments revealed that Bgp1 may be involved in a number of specific embryonic processes. Immunoblot analysis of Bgp1 deletion mutant proteins indicated that distinguishable epitopes of the molecule were preferentially identified by the three Bgp1 antibodies used in this study. This distinction is supported by our immunolocalization studies during mouse embryogenesis in which the three antibodies revealed specific patterns of Bgp1 expression. Bgp1 is not expressed in early post‐implantation embryos (7.5 dpc), but is found in the placenta and extra‐embryonic tissues (decidual endothelial cells, giant trophoblasts, yolk sac visceral endoderm, and endometrial glands) at this time. The primitive gut epithelium and surface ectoderm were the first embryonic tissues to express Bgp1. Significant Bgp1 expression was also observed later during epithelial‐mesenchymal interactions (skin, meninges, lung, kidney, salivary glands, pancreas). A unique epitope of Bgp1, detectable by the monoclonal antibody CC1, was also associated with mesenchymal expression and was prominent during myogenesis (secondary myotube formation) at sites of terminal differentiation. These studies suggest multiple roles for isoforms and glycoforms of the Bgp1 proteins localized in specific sites during prenatal development.

Cardiac Failure in C5-Deficient A/J Mice after Candida albicans Infection

ABSTRACT The effect of a deficiency in the C5 component of complement on the pathophyisology of infection with the fungal pathogen Candida albicans was studied by using the A/J inbred mouse strain and the BcA17 congenic mouse strain. Acute infection caused by intravenous injection of C. albicans blastospores is associated with rapid fungal replication in the heart, brain, and, in particular, kidneys of C5-deficient mice. Histological studies and analysis of markers for tissue damage indicated that the heart is the organ that is most affected and that it ultimately fails in C5-deficient mice. In A/J and BcA17 mice, tissue damage is associated with (i) cellular infiltration in the heart, which is not seen in the kidney despite the higher fungal load in the latter organ, and (ii) a very strong inflammatory response, including elevated levels of many cytokines and chemokines. This results in cardiomyopathy, which is associated with elevated levels of creatine kinase and cardiac troponin I in the circulation. Damage to the cardiac muscle is associated with metabolic changes, including hypoglycemia, decreased lipid utilization resulting in elevated levels of cardiac triglycerides, and unproductive glucose utilization linked to a dramatic increase in the level of pyruvate dehydrogenase kinase 4 (Pdk4), a negative regulator of the pyruvate dehydrogenase complex.

Expression of the Growth Factor Progranulin in Endothelial Cells Influences Growth and Development of Blood Vessels: A Novel Mouse Model

Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2–promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5–14.5) and later (E15.5–17.5) developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with subsequent effects in the formation of the mural cell layer and weakening of vessel integrity. These results demonstrate that overexpression of progranulin in endothelial cells influences normal angiogenesis in vivo.

Prep2: Cloning and expression of a new prep family member

We describe Prep2, a new murine homeobox‐containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain‐containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes.

Respiratory Distress and Neonatal Lethality in Mice Lacking Golgi α1,2-Mannosidase IB Involved in N-Glycan Maturation

There are three mammalian Golgi α1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man8-9GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi α1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on α1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The α1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.

A new microsurgical research model using Thiel-embalmed arteries and comparison of two suture techniques *

PURPOSE To assess the utility of the Thiel arterial model in microsurgical research, we compared interrupted horizontal mattress (HM) sutures to simple interrupted (SI) sutures in human vessels. METHODS A microsurgical set-up using an operating microscope and Thiel-embalmed arteries was used to practice ten SI and HM anastomoses. Vessel patency, leak and stricture were evaluated using angiography, and vessel wall architecture was evaluated using light microscopy and scanning electron microscopy (SEM). The technique speed was also assessed. RESULTS We have successfully evaluated all outcomes. All anastomoses were patent. The stricture rate was higher with HM than with SI (60% vs. 35% surface area reduction). Three minor leaks occurred with HM sutures versus one with SI sutures. Edges were evenly everted without any intimal flaps with HM compared to SI. The anastomoses were performed faster using HM than SI sutures (7:58 min vs. 12:41 min, respectively). CONCLUSION This is the first study to evaluate the feasibility of a Thiel-embalmed artery model for research purposes. The HM microvascular suture is a promising technique that requires further in vivo validation.