Study of interactions between DNA and aflatoxin B1 using electrochemical and fluorescence methods

In this study, a carbon paste electrode modified with N-butylpyridinium hexafluorophosphate (BPPF₆) ionic liquid and DNA was introduced as an electrochemical biosensor to study the interaction between DNA and aflatoxin B1 molecules. For this purpose, variations in oxidation peak current of guanine in various concentrations of aflatoxin B1 were measured by using the differential pulse voltammetry (DPV) method. According to this study, the binding constant of DNA–aflatoxin B1 was found to be 3.5 × 10⁶ M⁻¹. This modified electrode was also used for determination of low concentrations of aflatoxin B1 by using differential pulse voltammetry. A linear dynamic range from 8.00 × 10⁻⁸ to 5.91 × 10⁻⁷ M and a limit of detection of 2.00 × 10⁻⁸ M resulted from DPV measurements. To confirm our results, a fluorescence study was also performed. It resulted in a binding constant of 2.8 × 10⁶ M⁻¹, which is in good agreement with that obtained from electrochemical study.     

Proprotein convertase PC7 enhances the activation of the EGF receptor pathway through processing of the EGF precursor

Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific, it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs), only the membrane-bound PC7, the most ancient and conserved basic amino acid-specific PC family member, induces the processing of pro-EGF into an ～115-kDa transmembrane form (EGF-115) at an unusual VHPR(290)↓A motif. Because site-directed mutagenesis revealed that Arg(290) is not critical, the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors, which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation, as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus, PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.     

The spectrum of median craniofacial dysplasia

Given the multiple permutations in craniofacial malformations, classification of median craniofacial dysplasia or midline Tessier no. 0 to 14 clefts has been difficult and disjointed. In this review, the authors present a summary of normal embryology, prior terminology, and their proposed new classification system. Median craniofacial dysplasia has tissue agenesis and holoprosencephaly at one end (the hypoplasias), frontonasal hyperplasia and excessive tissue (the hyperplasias) at the other end, and abnormal splitting or clefting and normal tissue volume (dysraphia) occupying the middle portion of the spectrum. These three distinct subclassifications have different forms of anomalies within their groups.     

Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1

SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions.     

Unique cell wall abnormalities in the putative phosphoinositide phosphatase mutant <Emphasis Type="Italic">AtSAC9</Emphasis>

SAC9 is a putative phosphoinositide phosphatase in Arabidopsis thaliana involved in phosphoinositide signaling. sac9-1 plants have a constitutively stressed phenotype with shorter roots which notably accumulate phosphatidylinositol 4,5-bisphosphate and its hydrolysis product inositol trisphosphate. We investigated the primary roots of sac9-1 seedlings at the cytological and ultrastructural level to determine the structural basis for this altered growth. Despite the normal appearance of organelles and cytoplasmic elements, our studies reveal extreme abnormalities of cell wall and membrane structures in sac9-1 primary root cells, regardless of cell type, position within the meristematic area, and plane of section. Cell wall material was deposited locally and in a range of abnormal shapes, sometimes completely fragmenting the cell. Simple protuberances, broad flanges, diffuse patches, elaborate folds, irregular loops and other complex three-dimensional structures were found to extend randomly from the pre-existing cell wall. Abundant vesicles and excessive membrane material were associated with these irregular wall structures. We argue that a perturbed phosphoinositide metabolism most likely induces these observed abnormalities and hypothesize that a disorganized cytoskeleton and excessive membrane trafficking mediate the cell wall defects.     

The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.     

Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference

Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.