RNAi-Mediated Knock-Down of Arylamine N-acetyltransferase-1 Expression Induces E-cadherin Up-Regulation and Cell-Cell Contact Growth Inhibition

Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics

Identification and Characterization of RcMADS1, an AGL24 Ortholog from the Holoparasitic Plant Rafflesia cantleyi Solms-Laubach (Rafflesiaceae)

10.1371/journal.pone.0067243PLoS ONE86-POLN

Somatic mutations of CADM1 in aldosterone-producing adenomas and gap junction-dependent regulation of aldosterone production

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production

A chromosome conformation capture ordered sequence of the barley genome

201

Holoprosencephaly

Holoprosencephaly (HPE) is a complex brain malformation resulting from incomplete cleavage of the prosencephalon, occurring between the 18th and the 28th day of gestation and affecting both the forebrain and the face. It is estimated to occur in 1/16,000 live births and 1/250 conceptuses. Three ranges of increasing severity are described: lobar, semi-lobar and alobar HPE. Another milder subtype of HPE called middle interhemispheric variant (MIHF) or syntelencephaly is also reported. In most of the cases, facial anomalies are observed in HPE, like cyclopia, proboscis, median or bilateral cleft lip/palate in severe forms, ocular hypotelorism or solitary median maxillary central incisor in minor forms. These latter midline defects can occur without the cerebral malformations and then are called microforms. Children with HPE have many medical problems: developmental delay and feeding difficulties, epilepsy, instability of temperature, heart rate and respiration. Endocrine disorders like diabetes insipidus, adrenal hypoplasia, hypogonadism, thyroid hypoplasia and growth hormone deficiency are frequent. To date, seven genes have been positively implicated in HPE: Sonic hedgehog (SHH), ZIC2, SIX3, TGIF, PTCH, GLI2 and TDGF1. A molecular diagnosis can be performed by gene sequencing and allele quantification for the four main genes SHH, ZIC2, SIX3 and TGIF. Major rearrangements of the subtelomeres can also be identified by multiplex ligation-dependent probe amplification (MLPA). Nevertheless, in about 70% of cases, the molecular basis of the disease remains unknown, suggesting the existence of several other candidate genes or environmental factors. Consequently, a "multiple-hit hypothesis" of genetic and/or environmental factors (like maternal diabetes) has been proposed to account for the extreme clinical variability. In a practical approach, prenatal diagnosis is based on ultrasound and magnetic resonance imaging (MRI) rather than on molecular diagnosis. Treatment is symptomatic and supportive, and requires a multidisciplinary management. Child outcome depends on the HPE severity and the medical and neurological complications associated. Severely affected children have a very poor prognosis. Mildly affected children may exhibit few symptoms and may live a normal life

From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase : impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer