EpCAM: Structure and function in health and disease

Injection of tumor cells in mice more than 30 years ago resulted in the discovery of an epithelial antigen, later defined as a cell adhesion molecule (EpCAM). Although EpCAM has since evoked significant interest as a target in cancer therapy, mechanistic insights on the functions of this glycoprotein have been emerging only very recently. This may have been caused by the multitude of functions attributed to the glycoprotein, its localization at different subcellular sites and complex posttranslational modifications. Here, we review how EpCAM modifies cell–cell contact adhesion strength and tissue plasticity, and how it regulates cell proliferation and differentiation. Major knowledge derived from human diseases will be highlighted: Mutant EpCAM that is absent from the cell surface leads to fatal intestinal abnormalities (congenital tufting enteropathy). EpCAM-mediated cell proliferation in cancer may result from signaling (i) via regulated intramembrane proteolysis and/or (ii) the localization and association with binding partners in specialized membrane microdomains. New insight in EpCAM signaling will help to develop optimized cancer therapies and open new avenues in the field of regenerative medicine.     

Studies on the effect of reagent and protein charges on reactivity of the β93 sulfhydryl group of human hemoglobin using selected mutations

The reactivity of the β93 sulfhydryl (SH) group of human oxyhemoglobins with the negatively charged 5,5′-dithiobis(2,2′-nitrobenzoate) and the uncharged 2,2′-dithiodipyridine was determined as a function of pH. Selected mutant hemoglobins having increased oxygen affinity and having residue substitutions altering charge near the SH group (Wood, Malmö, Yakima, Kempsey, Andrew-Minneapolis, Osler, and Chesapeake) were compared to hemoglobin (Hb) A. Although both reagents reacted with GSH at the same rate and with the same enthalpies of activation, the rates with Hb were different and the difference showed a pronounced pH dependence. The charged reagent was sensitive to charges near the SH group; a positive charge increased the rate and a negative one decreased the rate. The uncharged reagent which reacted with Hb A with activation enthalpies similar to those for GSH was insensitive to neighboring charges, but was sensitive to tertiary and quaternary structural changes. The rates obtained with the latter reagent did not correlate with oxygen affinity. The evolutionary aspects of the β93 cysteine in relation to structure and function are reviewed.     

Synthesis of norbornanepentols: analogues of inositols

The synthesis of endo-5,6-exo-2,3-syn-7-norbornanepentol (5), endo-5-exo-2,3,6-syn-7-norbornanepentol (14), and 7-exo-2,3,5,6-norbornanepentol (16) are described. cis-Hydroxylation of 7-tert-butoxynorbornadiene (1) gave the exo-diol 2, endo-diol 3, and tetrol 4. The latter was deprotected to give pentol 5. Oxidation of alkene 6 afforded diacid 7 and two minor products: the exo-diol 8 and alpha-hydroxyketone 9. cis-Hydroxylation of 6 gave the endo- and exo-diols 10 and 8. Acetalation of 8 furnished the bis(dioxolane) 11. Reduction of ketone 9 gave the trans-diol 12. Deblocking of 8 and 12 led the tetrol 15 or pentols 16 and 14. The structure of tetrol 4 was confirmed by X-ray diffraction. Compounds 4, 5 and 16 were devoid of antitumor or antiviral activity.     

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.     

Seasonal cycles of plasma testosterone and cortisol in the wild rabbit (Oryctolagus cuniculus algirus Loche) of Zembra Island, studied in its natural biotope and in captivity

Annual variations in plasma testosterone and cortisol levels were determined in male wild rabbits caught monthly in their natural biotope (Zembra island). For comparison, a group of warrens was hold in semi-captivity close to Tunis. In both conditions plasma testosterone concentrations were low from January to September and peak values were observed in October. Captive warrens exhibited a clear cut annual rhythm in plasma cortisol levels also and ether stress resulted in a very large increase of cortisolemia. In rabbits caught in ther habitat however, cortisol concentrations were always extremely high throughout the year, probably due to stressing conditions. Interestingly, wild rabbits of Zembra island appear to subsist from a very remote past without any contact with other strains. As to Zembra, it is a hardly get-at-able, uninhabited island which is a part of a strictly protected natural reserve and constitutes a fairly useful and valuable ecosystem.     

The molecular machinery of germ line specification

Germ cells occupy a unique position in animal reproduction, development, and evolution. In sexually reproducing animals, only they can produce gametes and contribute genetically to subsequent generations. Nonetheless, germ line specification during embryogenesis is conceptually the same as the specification of any somatic cell type: germ cells must activate a specific gene regulatory network in order to differentiate and go through gametogenesis. While many genes with critical roles in the germ line have been characterized with respect to expression pattern and genetic interactions, it is the molecular interactions of the relevant gene products that are ultimately responsible for germ cell differentiation. This review summarizes the current state of knowledge on the molecular functions and biochemical connections between germ line gene products. We find that homologous genes often interact physically with the same conserved molecular partners across the metazoans. We also point out cases of nonhomologous genes from different species whose gene products play analogous biological roles in the germ line. We suggest a preliminary molecular definition of an ancestral “pluripotency module” that could have been modified during metazoan evolution to become specific to the germ line. Mol. Reprod. Dev. 77: 3–18, 2010. © 2009 Wiley‐Liss, Inc.     

Split Nano luciferase complementation for probing protein‐protein interactions in plant cells

Deciphering protein‐protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual‐use system enabling the detection of PPIs through the Nluc‐based SLC and co‐immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA‐ or FLAG‐tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium‐transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc‐based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.     

Characterization of Polar and Non‐Polar Compounds of House Edible Bird's Nest (EBN) from Johor,Malaysia

This work investigated the polar (PC: protein, amino acid and metabolite) and non‐polar (NPC: fatty acid) compounds and bioactivity characteristics of the EBN harvested from the state of Johor in Malaysia. The electrophoretic gels exhibited 15 protein bands (16–173 kD) with unique protein profile. Amino acids analysis by AccQ?Tag method revealed 18 types of amino acids in EBN. Metabolite profiling was performed using High‐Performance Liquid Chromatography coupled with Quadrupole Time‐of‐Flight Mass Spectrometer (HPLC‐QTOF/MS) technique and a total of 54 compounds belonging to different groups were detected and identified. These findings help to uncover the relation of therapeutic activity of EBN. The EBN was further extracted with AcOEt and BuOH. The AcOEt extract was fractionated into three fractions (F₁?F₃), and the high triglyceride content in F₂ was verified by gC‐FID. The three groups of fatty acids discovered in EBN are 48.43 % of poly‐unsaturated (PUFA), 25.35 % of saturated fatty acids (SFA) and 24.74 % of mono‐unsaturated fat (MUFA). This is the first time to report results ofEBN, BuOH, and AcOEt extracts and of fraction F₂ (TEBN) on their analysis for their antioxidant activities by DPPH, ABTS and catalase assay and for their paraoxonase and anti‐tyrosinase activities. The results showed that TEBN exhibited the significant bioactivity in all assays. These findings suggest that TEBN is a good source for natural bioactive compounds in promoting body vigor. Current work widened the content of EBN especially on the triglyceride and also marked the content of specific location (Johor, Malaysia) of EBN origin.