Genetic Analysis of the First 4 Patients with β-Ureidopropionase Deficiency

β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyses the irreversible hydrolysis of N-carbamyl-ß-aminoisobutyric acid or N-carbamyl-ß-alanine to β-aminoisobutyric acid or ß-alanine, ammonia, and CO₂. Analysis of the β-ureidopropionase gene (UPB1) of the first 4 patients presenting with a complete enzyme deficiency, revealed the presence of 2 splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). RT-PCR analysis of the complete β-ureidopropionase cDNA suggested that both splice-site mutations lead to a variety of alternative splice variants, with deletions of a single or several exons. The alanine at position 85 was not conserved in other eukaryotic β-ureidopropionase protein sequences.     

Resolvin D1 inhibits TGF-β1-induced epithelial mesenchymal transition of A549 lung cancer cells via lipoxin A4 receptor/formyl peptide receptor 2 and GPR32

Epithelial-mesenchymal-transition (EMT) is a key event for tumor cells to initiate metastasis which lead to switching of E-cadherin to N-cadherin. Resolvins are known to promote the resolution of inflammation and phagocytosis of macrophages. However, the role of resolvins in EMT of cancer is not known. Therefore, we examined the effects of resolvins on transforming growth factor, beta 1 (TGF-β1)-induced EMT. Expression of E-cadherin and N-cadherin in A549 lung cancer cells was evaluated by Western blot and confocal microscopy. Involvement of lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) was examined by gene silencing. TGF-β1 induced expression of N-cadherin in A549 lung cancer cells, and resolvin D1 and D2 inhibited the expression of N-cadherin at low concentrations (1–100 nM). Resolvin D1 and D2 also suppressed the expression of zinc finger E-box binding homeobox 1 (ZEB1). The effects of resolvin D1 and D2 were confirmed in other lung cancer cell lines such as H838, H1299, and H1703. Resolvin D1 and D2 did not affect the proliferation of A549 lung cancer cells. Resolvin D1 and D2 also suppressed the TGF-β1-induced morphological change. Resolvin D1 and D2 also inhibited the TGF-β1-induced migration and invasion of A549 cells. Resolvin D1 is known to act via ALX/FPR2 and GPR32. Thus, we examined the involvement of ALX/FPR2 and GPR32 in the suppressive effects of resolvin D1 on TGF-β1-induced EMT of A549 cells. Gene silencing of ALX/FPR2 and GPR32 blocked the action of resolvin D1. Overexpression of ALX/FPR2 or GPR32 increased the effects of resolvin D1. These results suggest that resolvin D1 inhibited TGF-β1-induced EMT via ALX/FPR2 and GPR32 by reducing the expression of ZEB1.     

Eosinophil Adhesion and Maturation is Modulated by Laminin

Eosinophils (Eo) participate in the inflammatory response to parasites, allergins, toxins, and epitopes recognized by autoimmune antibodies. Nonetheless, little attention has heretofore been paid to the interactions of Eo with extracellular matrix (ECM) proteins during their migration through the subendothelial basement membrane and into the surrounding tissue. Therefore, we have studied the adhesion of Eo to specific ECM proteins and the effect of this adhesion on Eo viability and maturation. Control Eo (from normal donors) adhere no better to substrates coated with laminin (LM), fibronectin (FN), cytotactin (CT), or collagen types I or IV (Col IV) than they do to human serum albumin coated substrates. In contrast, Eo activated in vitro with IL-5 or in vivo in patients with eosinophilia bind well to LM, FN and Col IV. LM is by far the most avid ligand among these molecules. For example, 43% of input cells bind to a substrate bearing 200 fmol/cm² of LM; a similar level of adhesion to FN requires 30 times as much adsorbed protein. Antibody inhibition experiments suggest that the αβ1 integrin heterodimer is the predominant LM receptor on these cells. Flow cytometry showed similar levels of these subunits on control and activated Eo, suggesting that Eo adhesion to LM is not regulated simply by cell surface integrin concentration. The effects of ECM proteins on Eo behavior were also examined. A LM-coated substrate (with no added cytokine) was found to be almost as effective as IL-5 in maintaining Eo viability while an equally adhesive FN-coated substrate had much less effect. Normally, even in the presence of 10% serum, no Eo survive a 5-day incubation in vitro unless IL-3, IL-5, or GM-CSF is added to the medium. Conditions that inhibit adhesion to LM (anti-integrin antibodies in the medium or CT on the substrate) and certain anti-cy-tokine antibodies inhibited the promotion of Eo viability by LM. During incubation on LM, Eo become hypodense, as they do in the presence of IL-5, indicating that they have become activated. These observations suggest that the interactions of Eo and ECM proteins may be important both for their potential to direct Eo migration and for their ability to regulate Eo viability, cytokine production, and maturation.     

Distribution of αvβT3, αvβTB5 Integrins and the Integrin Associated Protein — IAP (CD47) in Human Glomerular Diseases

The av integrins present on the membrane of numerous cells, mediate attachment to matrix proteins, cell proliferation, migration and survival. We studied the expression of av integrins and CD47 (a 03 chain integrin associated protein) in various forms of glomerulonephritis (GN) characterized by mesangial proliferation and/or increased mesangial matrix. In normal glomeruli, epithelial cells expressed αvβT3, αvβT5 and CD47; endothelial cells expressed α5βT1 and CD47; mesangial cells expressed αvβT5, CD47, and to a less extent αvβT3. In acute post infectious GN (APIGN), membranoproliferative GN (MPGN) and diabetic nephropathy (DN), we observed that the βT3 chain, normally expressed by mesangial cells, was not detectable in the mesangium while its expression by epithelial cells was not modified. Parallel to the disappearance of αvβT3, the CD47 expression was decreased on the mesangial cells in MPGN, APIGN and DN. The expression of αvβT5 was clearly increased on podocytes and on proliferating mesangial cells in APIGN. By contrast, the mesangial expression of αvβT5 normal or decreased in DN. The α5 chain of integrin, absent on normal mesangial cell, was expressed on proliferating mesangial cells in MPGN and APIGN.

Thus, we observed modifications of avp3 and avp5 expression during human GN. The modulations of αvβT3 and αvβT5 expression differed according to the different glomerular cell types and were not parallel in glomerular cells: avp3 was decreased (and αvβT5 unchanged) on proliferating mesangial cells and αvβT5 was increased (and αvβT3 unchanged) in podocytes. This may reflect the existence of two distinct regulatory pathways.     

INTERFERENTIAL ELECTRIC FIELDS REVEAL NONUNIFORM EFFECTS ON PROLIFERATION AND hCG SECRETION IN JAr CELLS

The effect of low frequency electromagnetic fields on changes in intracellular cAMP concentrations was investigated in the frequency range 10–100 Hz using a choriocarcinoma cell line. JAr cells significantly reduce proliferation and increase β-hCG secretion upon dibutyryl cAMP and forskolin treatment after 10 days of culturing. Choriocarcinoma cells exposed to a modulation frequency of 10 Hz for 5 min change their intracellular cAMP level significantly to higher as well as to lower concentrations in half of the experimental series, respectively. At frequencies of 70 and 100 Hz levels, half of the experimental series revealed a significant decrease in cAMP levels. Long term exposure at 100 Hz for 10 days leads to a significant reduction in proliferation but not in β-hCG secretion. These results point to a modulatory effect of low frequency electromagnetic fields on intracellular cAMP levels which are dependent on the frequency window. The reduced proliferation after long term exposure at the frequency of 100 Hz, which lowers cAMP levels, is discussed.     

CaMKKβ-AMPKα2 signaling contributes to mitotic Golgi fragmentation and the G2/M transition in mammalian cells

Before a cell enters mitosis, the Golgi apparatus undergoes extensive fragmentation. This is required for the correct partitioning of the Golgi apparatus into daughter cells, and inhibition of this process leads to cell cycle arrest in G2 phase. AMP-activated protein kinase (AMPK) plays critical roles in regulating growth and reprogramming metabolism. Recent studies have suggested that AMPK promotes mitotic progression and Golgi disassembly, and that this seems independent of the cellular energy status. However, the molecular mechanism underlying these events is not well understood. Here, we show that both treatment with compound C and depletion of AMPKα2 (but not AMPKα1) delays the G2/M transition in synchronized HeLa cells, as evidenced by flow cytometry and mitotic index analysis. Furthermore, knockdown of AMPKα2 specifically delays further fragmentation of isolated Golgi stacks. Interestingly, pAMPKα^Thr172 signals transiently appear in the perinuclear region of late G2/early prophase cells, partially co-localizing with the Golgi matrix protein, GM-130. These Golgi pAMPKα^Thr172 signals were also specifically abolished by AMPKα2 knockdown, indicating specific spatio-temporal activation of AMPKα2 at Golgi complex during late G2/early prophases. We also found that the specific CaMKKβ inhibitor, STO-609, reduces the pAMPKα ^Thr172 signals in the perinuclear region of G2 phase cells and delays mitotic Golgi fragmentation. Taken together, these data suggest that AMPKα2 is the major catalytic subunit of AMPKα which regulates Golgi fragmentation and G2/M transition, and that the CaMKKβ activates AMPKα2 during late G2 phase.     

Powerful Drosophila screens that paved the wingless pathway

The Wnt/Wingless (Wg) signaling cascade controls a number of biological processes in animal development and adult life; aberrant Wnt/Wg signaling can cause diseases. In the 1980s genes were discovered that encode core Wnt/Wg pathway components: their mutant phenotypes were similar and an outline of a signaling cascade emerged. Over the years our knowledge of this important signaling system increased and more components were uncovered that are instrumental for Wnt/Wg secretion and transduction. Here we provide an overview of these discoveries, the technologies involved, with a particular focus on the important role Drosophila screens played in this process.     

The role of adherens junctions in the developing neocortex

The disproportional enlargement of the neocortex through evolution has been instrumental in the success of vertebrates, in particular mammals. The neocortex is a multilayered sheet of neurons generated from a simple proliferative neuroepithelium through a myriad of mechanisms with substantial evolutionary conservation. This developing neuroepithelium is populated by progenitors that can generate additional progenitors as well as post-mitotic neurons. Subtle alterations in the production of progenitors vs. differentiated cells during development can result in dramatic differences in neocortical size. This review article will examine how cadherin adhesion proteins, in particular α-catenin and N-cadherin, function in regulating the neural progenitor microenvironment, cell proliferation, and differentiation in cortical development.     

Protein Misfolding and Aggregation in Ageing and Disease

Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as ‘conformational diseases’ and include neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.     

Metallo-β-lactamase: Inhibitors and reporter substrates

Metallo-β-lactamases represent an emerging clinical threat due to their ability to render ineffective an entire class of antibiotics. Accordingly, this family of enzymes has been suggested as an attractive target for drug design. Progress toward developing effective inhibitors as well as the development of reporter substrates is reviewed. Inhibitors are classified into six classes and known binding interactions with metallo-β-lactamases are summarized. The development of chromogenic and fluorogenic reporter substrates is also reviewed with respect to current and prospective applications to future inhibitor and diagnostic discovery, mechanistic studies, and biological imaging. Despite progress in molecular probe development, the sequence and structural diversity within the metallo-β-lactamase family continue to present substantial hurdles for rational ligand design.