The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer

In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs)

Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane

Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues

Molecular mechanism of HMGA1 deregulation in human neuroblastoma

Very soon after their original identification in HeLa cells in 1983, HMGA proteins appeared as interesting cancer-related molecules. Indeed, they were immediately noted as a sub-class of High Mobility Group proteins induced in fibroblast or epithelial cells transformed with sarcoma viruses. After more than 20 years, the association between HMGA protein expressions and cellular transformation has been largely confirmed and HMGA are among the most widely expressed cancer-associated proteins. Nevertheless, their functional contribution to tumour development and progression is far from being completely understood. Furthermore, although HMGA1 expression has been reported to be inducible by a number of factors and circumstances, the question of how their expression is deregulated in cancer is even less clear and somehow has been ignored from most researchers. An active AP1 site is the only characterized element of the HMGA1 human promoter, that remains a rather complicated and unexplored source of information to answer this question. Following the indication that c-Myc might bind and activate the mouse HMGA1 gene promoter, we have demonstrated that HMGA1 is a new target for MYCN in human neuroblastomas. In this report, we overview part of the current information on HMGA I and focus our attention on the analysis of its human promoter. (C) 2005 Elsevier Ireland Ltd. All rights reserved

"While there is p57, there is hope." The past and the present of diagnosis in first trimester abortions: Diagnostic dilemmas and algorithmic approaches. A review

Distinction of hydatidiform moles (HM) from non-molar (NM) specimens and subclassification of HM as complete hydatidiform mole (CHM) versus partial hydatidiform mole (PHM) are important for clinical practice and investigational studies. The issue of diagnostic reproducibility is still unsolved, the lack of diagnostic accuracy based on morphology is substantial with an important interobserver variability, even between experienced gynecologic pathologists. Many ancillary techniques have been investigated in the last years to refine HM diagnosis. p57 (a paternally imprinted, maternally expressed gene) immunohistochemistry, based on the unique genetics of CHM (purely androgenetic), PHM (diandric triploid), and NM specimens (biparental, with allelic balance) can identify CHMs, which lack p57 expression because of a lack of maternal DNA. However, although its role in HM diagnosis is pivotal, it does not allow the distinction of PHM from NM specimens, both of which express p57 due to the presence of maternal DNA. Molecular genotyping, which compares villous and decidual DNA patterns to determine the parental source and ratios of polymorphic alleles, distinguishes purely androgenetic CHM from diandric triploid PHM, and both of these from NM specimens. Beyond the claim of establishing a "diagnostic truth", exceptions and peculiar genetic scenarios in the origin of rare CHM and PHM should be kept in mind when approaching any ancillary technique. An algorithmic approach, even in settings with limited resources, can help the pathologists in the diagnostic dilemma of diagnosis of first trimester abortions

Atomic force microscopy shows connexin26 hemichannel clustering in purified membrane fragments.

Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell-cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels

Gestational Diabetes—Placental Expression of Human Equilibrative Nucleoside Transporter 1 (hENT1): Is Delayed Villous Maturation an Adaptive Pattern?

Gestational diabetes mellitus (GDM) is a metabolic disease that can affect placental villous maturation and villous vascularity. The main effects of GDM on placental growth are a delay of villous maturation (DVM) and decreased formation of vasculo-syncytial membranes (VSM). Human equilibrative nucleoside transporter-1 (hENT1) is an adenosine transporter expressed in the human umbilical vein endothelial cells (HUVEC) and human placental microvascular endothelium cells (hPMEC). Its role is crucial in maintaining physiological fetal adenosine levels during pregnancy, and its reduction has been described in GDM. Twenty-four placentas from pregnancies with a confirmed diagnosis of GDMd and twenty-four matched non-GDM placentas (controls) were retrospectively analyzed to investigate the immunohistochemical expression of hENT1 in HUVEC and hPMEC. The study included the quantitative evaluation of VSM/mm2 in placental tissue and the immunohistochemical quantitative evaluation of Ki-67, PHH3, and p57 in villous trophoblast. hENT1 expression was higher in all the vascular districts of the control cases compared to the GDMd placentas (p 2 were lower in the GDMd cases, while the Ki-67, PHH3, and p57 were higher when compared to the control cases. To our knowledge, this is the first report of hENT1 expression in the human placentas of GDM patients. The absence/low expression of hENT1 in all the GDMd patients may indicate a potential role in microvascular adaptative mechanisms. The trophoblasts’ proliferative/antiapoptotic pattern (high Ki-67, high PHH3, and high p57 count) may explain the statistically significant lower number of VSM/mm2 found in the GDMd cases

Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1.

Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264-275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence showed colocalization of Cx43, drebrin, and F-actin in astrocytes and Vero cells membrane, indicating that Cx43 forms a submembrane protein complex with cytoskeletal and scaffolding proteins. The co-IP data suggest that Cx43 indirectly interacts with F-actin through drebrin. Along with the known interaction of the Cx43-CT with ZO-1 and tubulin, the data presented here for drebrin indicate non-overlapping and separated binding sites for all three proteins for which simultaneous binding could be important in regulating cytoskeleton rearrangements, especially for neuronal migration during brain development