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OBJECTIVE: Smad-interacting protein-1 (Sip1/ZEB2) is a transcriptional repressor of the telomerase reverse transcriptase catalytic subunit (Tert) and has recently been identified as a key regulator of embryonic cell fate with a phenotypic effect similar, in our opinion, to that reported for nitric oxide (NO). Remarkably, SIP1/ZEB2 is a known target of the microRNA 200 (miR-200) family. In this light, we postulated that Sip1/ZEB2 and the miR-200 family could play a role during the NO-dependent differentiation of mES. METHODS AND RESULTS: The results of the present study show that Sip1/ZEB2 expression is downregulated during the NO-dependent expression of mesendoderm and early cardiovascular precursor markers, including Flk1 and CXCR4 in mES. Coincidently, members of the miR-200 family, namely miR-429, -200a, -200b, and -200c, were transcriptionally induced in parallel to mouse Tert. This regulation occurred at the level of chromatin. Remarkably, miR-429/miR-200a overexpression or Sip1/ZEB2 knockdown by short hairpin RNA interference elicited a gene expression pattern similar to that of NO regardless of the presence of leukemia inhibitory factor. CONCLUSIONS: These results are the first demonstrating that the miR-200 family and Sip1/ZEB2 transcription factor are regulated by NO, indicating an unprecedented molecular circuitry important for telomerase regulation and early differentiation of mES.

Several studies have demonstrated that exposure to both acute and chronic aversive stimuli can affect neural activity in different brain areas. In particular it has been shown that stressful events can induce not only short-term changes in neural transmission and gene regulation, but also long-term changes that can lead to structural modification. In this study we investigated, in CD1 mice, the effects of single or repeated exposures to restraint stress (2h for 1 or 5 consecutive days) in the frontal cortex on a crucial class of gene expression regulators, the microRNAs (miRs).First we performed a microarray profiling on RNA extracted from the frontal cortex of mice exposed to acute or repeated restraint stress. The results indicated a prominent increase in the expression levels of different miRs after acute stress while only minor changes were observed after repeated restraint. The Northern blot analysis on selected miRs confirmed an increase after acute restraint for let-7a, miR-9 and miR 26-a/b. Finally, Northern blot analysis of the selected miRs on RNA extracted from the hippocampus of stressed mice demonstrated that such changes were region specific, as no differences were observed in the hippocampus. These data suggest that control of mRNA translation through miRs is an additional mechanism by which stressful events regulates protein expression in the frontal cortex.

Small non coding RNAs are a group of very different RNA molecules, present in virtually all cells, with a wide spectrum of regulatory functions which include RNA modification and regulation of protein synthesis. They have been isolated and characterized in all organisms and tissues, from Archaeobacteria to mammals. In mammalian brain there are a number of these small molecules, which are involved in neuronal differentiation as well as, possibly, in learning and memory. In this manuscript, we analyze the present knowledge about the function of the most important groups of small non-coding RNA present in brain: small nucleolar RNAs, small cytoplasmic RNAs, and microRNAs. The last ones, in particular, appear to be critical for dictating neuronal cell identity during development and to play an important role in neurite growth, synaptic development and neuronal plasticity.

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-mm diol-silica particles. The CSP mixed with 5-mm amino silica particles (3:1) was packed into 75-mm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor R 52.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12–20 min).

Enantiomeric separation of some selected acidic compounds of pharmaceutical interest belonging to the group of non-steroidal anti-inflammatory drugs were separated by capillary electrochromatography employing silica based glycopeptide antibiotic stationary phases, namely vancomycin or a teicoplanin derivatives (Hepta-Tyr). The vancomycin stationary phase allowed to achieve the chiral resolution of some racemic studied compounds only using mobile phases containing ammonium formate at a relatively low pH 2.5–3.5 and acetonitrile. Employing the teicoplanin derivative stationary phase, good enantiomeric resolution was achieved eluting with mobile phases containing sodium phosphate pH 6–acetonitrile. Enantiomers were moved to the detector because a relatively high reversed electroosmotic flow (due to the positive charge of the stationary phase) and to the electrophoretic mobility of analytes.

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 mm diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 mm ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.

Different types of fused-silica capillaries of 75 mm inside diameter (ID) were packed, namely type A and B, and evaluated for the direct resolution of racemates of several basic compounds by enantioselective capillary electrochromatography (e-CEC). Type A was packed with a chiral stationary phase (CSP) containing teicoplanin (TE) mixed with silica microparticles (3:1 w/w) while type B contained only the TE-CSP. In both cases, particles of different sizes (3.5 and 5 mm ID) were employed. A polar-organic mobile phase containing methanol-acetonitrile (60–40% v/v and 0.05% w/v ammonium acetate was used. Several b-blockers (alprenolol, oxprenolol, metoprolol, pindolol, salbutamol, propranolol, atenolol, acebutolol) were baseline-enantioresolved with both capillary types, in very short times.

The separation of racemic derivatized amino acids (N-acetyl) in their enantiomers was achieved using capillary zone electrophoresis employing vancomycin as a chiral selector. Due to the strong absorption properties of the chiral selector at the low wavelengths used, the partial filling- counter current method was adopted in order to improve the method sensitivity. In the separation system experimented the chiral selector filled only a part of the capillary and, due to the appropriate selection of the pH, was moving in the opposite direction of the analytes keeping the detector free of absorbing compounds. The effect of several experimental parameters on enantioresolution of analytes was studied, e.g., vancomycin concentration (0-5 mM), pH of the background electrolyte (pH 4-7), capillary temperature (15-35 °C) and presence of an organic modifier into the run buffer (methanol or ethanol or n-propanol). N-acetyl glutamic acid, serine, cystine, tyrosine and proline were all baseline resolved in their enantiomers and the enantioresolution factor (Rs) increased by raising the vancomycin concentration. pH 4 allowed the baseline resolution of the 5 studied analytes in presence of 2.5 mM of chiral selector and an increase of pH caused a decrease of (Rs).

Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2'-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D' of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-L-methionine-binding region of Nop1p is responsible for the catalytic activity

The embryotoxicity of 4-chloro-2-methylphenoxyacetic acid (MCPA), an extensively used herbicide, has been evaluated and compared to that of phenol and chlorocresol (two common contaminants) with a bioassay that makes use of embryos of the amphibian Xenopus. The MCPA—Na salt used in the bioassay was purified by crystallization and acid-base purification methods, and the concentrations of phenol and chlorocresol were checked by high-performance liquid chromotography. The relationship between the concentration of the tested molecule and the outcomes (i.e., mortality and malformations) was investigated using different models (probit, logit, and complementary log-log). The resulting LC50s for MCPA, chlorocresol, and phenol are 3,607, 13, and 178 mg/L, respectively; the resulting LC10s are 1,526, 6, and 32 mg/L. It is evident that the real MCPA toxicity can be masked by the presence of contaminants much more toxic than the molecule studied. Moreover, our results show that the three chemicals do not present a high teratogenic risk. Growth retardation shows that MCPA, chlorocresol, and phenol are effective at concentrations as low as 2,000, 2.5, and 25 mg/L, respectively.

In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript.