Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.     

Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease

Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes. Here we show that missense, nonsense and frameshift mutations in SLC6A5 (ref. 8), encoding the presynaptic glycine transporter 2 (GlyT2), also cause hyperekplexia. Individuals with mutations in SLC6A5 present with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnea episodes. SLC6A5 mutations result in defective subcellular GlyT2 localization, decreased glycine uptake or both, with selected mutations affecting predicted glycine and Na+ binding sites.     

Claudin-17 forms tight junction channels with distinct anion selectivity

Barrier properties of tight junctions are determined by the claudin protein family. Many claudins seal this barrier, but others form paracellular channels. Among these, no claudins with general and clear-cut anion selectivity have yet been described, while for claudin-10a and claudin-4, only circumstantial or small anion selectivities have been shown. A claudin with unknown function and tissue distribution is claudin-17. We characterized claudin-17 by overexpression and knock-down in two renal cell lines. Overexpression in MDCK C7 cell layers caused a threefold increase in paracellular anion permeability and switched these cells from cation- to anion-selective. Knockdown in LLC-PK(1) cells indorsed the finding of claudin-17-based anion channels. Mutagenesis revealed that claudin-17 anion selectivity critically depends on a positive charge at position 65. Claudin-17 expression was found in two organs: marginal in brain but abundant in kidney, where expression was intense in proximal tubules and gradually decreased towards distal segments. As claudin-17 is predominantly expressed in proximal nephrons, which exhibit substantial, though molecularly not defined, paracellular chloride reabsorption, we suggest that claudin-17 has a unique physiological function in this process. In conclusion, claudin-17 forms channels within tight junctions with distinct anion preference.     

Effect of L-amino-ethyl-cysteine,a sulfur analogue of L-lysine,on virus miltiplication in mammalian cell cultures

Summary L-amino-ethyl-cysteine clearly inhibits replication of Mengovirus; another RNA-virus (vesicular stomatitis virus) is completely insensitive. Protein synthesis is not impaired, but no active viral RNA-polymerase is detected.This work was partly supported by grant No. 73.01407.44 115.0107 of Consiglio Nazionale delle Ricerche, Roma, Italy. The technical collaboration of P. Meo and G. Conciatori is gratefully acknowledged.     

Studies on the cytochemical localization of adenylate-cyclase activity inDugesia lugubris s.l.

Summary The localization of adenylate-cyclase activity inDugesia lugubris s.l. has been investigated cytochemically using 5-adenylyl-imidodiphosphate as substrate. The enzyme was localized in mucous gland cells, in rhabdite cells, in intercellular spaces and also in nerve endings of this planarian. The presence of adenylate-cyclase on the membrane suggests that it might mediate different stimulus-secretion coupling by increasing cyclic AMP synthesis in specialized areas of the planarian.Supported in part by a grant from Consiglio Nazionale delle Ricerche, Rome.Acknowledgments. This work was performed with the technical assistence of the laboratory of Electron Microscopy of the University of Perugia.     

Reciprocal crossover asymmetry and meiotic drive in a human recombination hot spot

Human DNA diversity arises ultimately from germline mutation that creates new haplotypes that can be reshuffled by meiotic recombination. Reciprocal crossover generates recombinant haplotypes but should not influence the frequencies of alleles in a population. We demonstrate crossover asymmetry at a recombination hot spot in the major histocompatibility complex, whereby reciprocal exchanges in sperm map to different locations in the hot spot. We identify a single-nucleotide polymorphism at the center of the hot spot and show that, when heterozygous, it seems sufficient to cause this asymmetry, apparently by influencing the efficiency of highly localized crossover initiation. As a consequence, crossovers in heterozygotes are accompanied by biased gene conversion, most likely occurring by gap repair, that can also affect nearby polymorphisms through repair of an extended gap. The result is substantial over-transmission of the recombination-suppressing allele and neighboring markers to crossover products. Computer simulations show that this meiotic drive, although weak at the population level, is sufficient to favor eventual fixation of the recombination-suppressing variant. These findings provide an explanation for the relatively uniform widths of human crossover hot spots and suggest that hot spots may be generally prone to extinction by meiotic drive.     

The effect of neuraminidase on the rabbit urinary interferon

Riassunto Proteine urinarie di coniglio contenenti interferone con una attività di 5000 U/mg sono state desializzate mediante neuraminidasi. La rapida e completa rimozione dell'acido sialico non si accompagna alla scomparsa della attività antivirale e pertanto, se l'interferone contiene acido sialico, quest'ultimo non ha un ruolo nell'attività biologica.     

Isoelectric fractionation of desialyzed interferon

Riassunto Proteine urinarie di coniglio contenenti interferone sono state desializzate mediante neuraminidasi e sono state separate simultaneamente ai controlli mediante elettroforesi su poliacrilammide con gradiente di pH. La rimozione dell'acido sialico produce una notevole modificazione del profilo elettroforetico con diminuzione dell'attività interferonica avente punti isoelettrici inferiori a pH 6.3. Il risultato indica che almeno in parte l'interferone contiene acido sialico.

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This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Roma, Gruppo Nazionale di Medicina Sperimentale.