Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P₂. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P₂ or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.     

Inhibition of Jack Bean Urease by N-(n-butyl)thiophosphorictriamide and N-(n-butyl)phosphorictriamide: Determination of the Inhibition Mechanism

N-(n-butyl)thiophosphorictriamide (NBPT) and its oxygen analogue N-(n-butyl)phosphorictriamide (NBPTO) were studied as inhibitors of jack bean urease. NBPTO was obtained by spontaneous conversion of NBPT into NBPTO. The conversion under laboratory conditions was slow and did not affect NBPT studies. The mechanisms of NBPT and NBPTO inhibition were determined by analysis of the reaction progress curves in the presence of different inhibitor concentrations. The obtained plots were time-dependent and characteristic of slow-binding inhibition. The effects of different concentration of NBPT and NBPTO on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships for a one-step enzyme-inhibitor interaction, qualified as mechanism A. The inhibition constants of urease by NBPT and NBPTO were found to be 0.15 μM and 2.1 nM, respectively. The inhibition constant for NBPT was also calculated by steady-state analysis and was found to be 0.13 μM. NBPTO was found to be a very strong inhibitor of urease in contrast to NBPT.     

Distinctive Subdomains in the Resorbing Surface of Osteoclasts

We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a ‘sealing zone’, encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but also to facilitate resorption by acting as a ‘functional sealing zone’.     

Sporadic Colorectal Cancer Development Shows Rejuvenescence Regarding Epithelial Proliferation and Apoptosis

Background and Aims

Sporadic colorectal cancer (CRC) development is a sequential process showing age-dependency, uncontrolled epithelial proliferation and decreased apoptosis. During juvenile growth cellular proliferation and apoptosis are well balanced, which may be perturbed upon aging. Our aim was to correlate proliferative and apoptotic activities in aging human colonic epithelium and colorectal cancer. We also tested the underlying molecular biology concerning the proliferation- and apoptosis-regulating gene expression alterations.

Materials and Methods

Colorectal biopsies from healthy children (n₁ = 14), healthy adults (n₂ = 10), adult adenomas (n₃ = 10) and CRCs (n₄ = 10) in adults were tested for Ki-67 immunohistochemistry and TUNEL apoptosis assay. Mitosis- and apoptosis-related gene expression was also studied in healthy children (n₁ = 6), adult (n₂ = 41) samples and in CRC (n₃ = 34) in HGU133plus2.0 microarray platform. Measured alterations were confirmed with RT-PCR both on dependent and independent sample sets (n₁ = 6, n₂ = 6, n₃ = 6).

Results

Mitotic index (MI) was significantly higher (p<0.05) in intact juvenile (MI = 0.33±0.06) and CRC samples (MI = 0.42±0.10) compared to healthy adult samples (MI = 0.15±0.06). In contrast, apoptotic index (AI) was decreased in children (0.13±0.06) and significantly lower in cancer (0.06±0.03) compared to healthy adult samples (0.17±0.05). Eight proliferation- (e.g. MKI67, CCNE1) and 11 apoptosis-associated genes (e.g. TNFSF10, IFI6) had altered mRNA expression both in the course of normal aging and carcinogenesis, mainly inducing proliferation and reducing apoptosis compared to healthy adults. Eight proliferation-associated genes including CCND1, CDK1, CDK6 and 26 apoptosis-regulating genes (e.g. SOCS3) were differently expressed between juvenile and cancer groups mostly supporting the pronounced cell growth in CRC.

Conclusion

Colorectal samples from children and CRC patients can be characterized by similarly increased proliferative and decreased apoptotic activities compared to healthy colonic samples from adults. Therefore, cell kinetic alterations during colorectal cancer development show uncontrolled rejuvenescence as opposed to the controlled cell growth in juvenile colonic epithelium.     

2-Substituted 4-hydroxybutanamides as potential inhibitors of γ-aminobutyric acid transporters mGAT1–mGAT4: Synthesis and biological evaluation

A series of 2-substituted 4-hydroxybutanamide derivatives has been synthesized by the aminolysis of appropriate 2-substituted dihydrofuran-2(3H)-one derivatives with various substituted benzylamines. The final compounds have been evaluated for their capability of inhibiting the GABA transport proteins GAT1-4 stably expressed in HEK-239 cell lines. The pIC₅₀ values determined were in the range 4.21–5.14. Two compounds (16a and 16d), which displayed the most interesting profiles in in vitro tests, have also been subjected to further preliminary behavioral studies, evaluating their antinociceptive activity in hot-plate, writhing, and formalin tests. Their influence on motor coordination has also been assessed.     

Comparative analysis of hydrogen-producing bacterial biofilms and granular sludge formed in continuous cultures of fermentative bacteria

A system for biohydrogen production was developed based on long-term continuous cultures grown on sugar beet molasses in packed bed reactors. In two separate cultures, consortia of fermentative bacteria developed as biofilms on granitic stones. In one of the cultures, a granular sludge was also formed. Metagenomic analysis of the microbial communities by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the overall biodiversity of the hydrogen-producing cultures was quite small. The stone biofilm from the culture without granular sludge was dominated by Clostridiaceae and heterolactic fermentation bacteria, mainly Leuconostocaeae. Representatives of the Leuconostocaeae and Enterobacteriaceae were dominant in both the granules and the stone biofilm formed in the granular sludge culture. The culture containing granular sludge produced hydrogen significantly more effectively than that containing only the stone biofilm: 5.43 vs. 2.8 mol H₂/mol sucrose from molasses, respectively. The speculations that lactic acid bacteria may favor hydrogen production are discussed.     

Synthesis, X-ray crystal structures and biomimetic and anticancer activities of novel copper(II)benzoate complexes incorporating 2-(4'-thiazolyl)benzimidazole (thiabendazole), 2-(2-pyridyl)benzimidazole and 1,10-phenanthroline as chelating nitrogen donor ligands

Cu(BZA)(2)(EtOH)(0.5) (1) was generated by the reaction of copper(II) hydroxide with benzoic acid (BZAH). [Cu(TBZH)(2)(BZA)](BZA).0.5TBZH.H(2)O (2) and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)].1.66EtOH (3) were obtained when 1 reacted with Thiabendazole (TBZH) and 2-(2-pyridyl)benzimidazole (2-PyBZIMH), respectively. [Cu(BZA)(2)(phen)(H(2)O)] (4) was isolated from the reaction of benzoic acid and 1,10-phenanthroline (phen) with copper(II)acetate dihydrate. Molecular structures of 2, 3 and 4 were determined crystallographically. 2 and 3 are hydrogen bonded dimers and trimers, respectively. The copper centres in complexes 2 and 3 are bis-chelate derivatives that have N(4)O ligation and their geometry is very similar being approximately square-pyramidal. However whereas in complex 2 both TBZH ligands are neutral in 3 one of the 2-PyBZIMH chelators is deprotonated on each copper. The structural results for 4 represent a re-examination of this crystallographically known compound for which no hydrogen atom coordinates have been previously reported. It crystallises as a hydrogen bonded dimmer and is a mono-chelate of phen with each copper centre possessing N(2)O(3) ligation and square pyramidal geometry. The catalase and superoxide dismutase (SOD) activities of the four complexes along with those of the known phenanthroline complexes [Cu(mal)(phen)(2)] and [Cu(phendione)(3)](ClO(4))(2) (malH(2)=malonic acid and phendione=1,10-phenanthroline-5,6-dione) were investigated. Complexes 1-4, the metal free ligands and a simple copper(II) salt were assessed for their cancer chemotherapeutic potential against the hepatocellular carcinoma (Hep-G(2)) and kidney adenocarcinoma (A-498) cell lines. TBZH, 2-PyBZIMH and benzoic acid when uncoordinated to a metal centre offer poor chemotherapeutic potential. copper(II) benzoate is significantly more active than the free acid. The bis-chelate derivatives [Cu(TBZH)(2)(BZA)](BZA).0.5TBZH.H(2)O (2) and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)].1.66EtOH (3) elicit a significant cytotoxic response to the cancer cell lines tested. Replacing TBZH and 2-PyBZIMH with phen to give [Cu(BZA)(2)(phen)(H(2)O)] (4) does not significantly increase the anti-cancer activity.     

Extracellular interactions between GluR2 and N-cadherin in spine regulation

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.     

Transition from chromatin bodies to linear chromosomes in nuclei of murine PreB cells synchronized in S phase

Chromatin structures and individual interphase chromosomes escaping nuclei of reversibly permeabilized cells were analyzed in a cell cycle-dependent manner. Cells were synchronized by counterflow centrifugal elutriation. Individual interphase chromosomes became visible as distinct fibrous chromatin bodies from mid-S-phase, turning to elongated chromosomes by the end of S phase. Major interphase chromosomal forms include (1) mid-S-phase chromatin bodies at 3.0 C-value, (2) elongated chromatin bodies later in mid-S-phase (3.25 C-value), (3) chromatin bodies with head and leg portions later in S phase (3.5 C-value), (4) supercoiled ribbons later in S phase seen as twisted prechromosomes (3.7 C-value), and (5) end-S-phase elongated, bent prechromosomal structures (3.9 C-value). The first karyotype analysis of the earliest forms of chromosomes referred to as chromatin bodies was performed.