microRNAs Modulate Spatial Memory in the Hippocampus and in the Ventral Striatum in a Region-Specific Manner.

MicroRNAs are endogenous, noncoding RNAs crucial for the post-transcriptional regulation of gene expression. Their role in spatial memory formation, however, is poorly explored. In this study, we analyzed learning-induced microRNA expression in the hippocampus and in the ventral striatum. Among miRNAs specifically downregulated by spatial training, we focused on the hippocampus-specific miR-324-5p and the ventral striatum-specific miR-24. In vivo overexpression of the two miRNAs demonstrated that miR-324-5p is able to impair memory if administered in the hippocampus but not in the ventral striatum, while the opposite is true for miR-24. Overall, these findings demonstrate a causal relationship between miRNA expression changes and spatial memory formation. Furthermore, they provide support for a regional dissociation in the post-transcriptional processes underlying spatial memory in the two brain structures analyzed.

Primary anorectal malignant melanoma: report of two cases.

Primary anorectal malignant melanoma is a fairly uncommon but highly malignant disease. It is sometimes mistaken for benign conditions such as hemorrhoids or rectal polyps. Here we describe two cases of primary malignant melanoma of the rectum: in one patient a wide local excision (WLE) was performed and in the other an abdominoperineal resection (APR), both with curative intent. Both patients developed systemic recurrences and died of their disease at 24 and 10 months, respectively. In conclusion, the prognosis of anorectal melanoma is poor, irrespective of surgical treatment. WLE is the first choice for primary anorectal melanoma, while APR should be reserved for those cases where complete transrectal tumor resection is technically impossible.

Processing of the intron-encoded U16 and U18 snoRNAs: the conserved C and D boxes control both the processing reaction and the stability of the mature snoRNA.

A novel class of small nucleolar RNAs (snoRNAs), encoded in introns of protein coding genes and originating from processing of their precursor molecules, has recently been described. The L1 ribosomal protein (r-protein) gene of Xenopus laevis and its human homologue contain two snoRNAs, U16 and U18. It has been shown that these snoRNAs are excised from their intron precursors by endonucleolytic cleavage and that their processing is alternative to splicing. Two sequences, internal to the snoRNA coding region, have been identified as indispensable for processing the conserved boxes C and D. Competition experiments have shown that these sequences interact with diffusible factors which can bind both the pre-mRNA and the mature U16 snoRNA. Fibrillarin, which is known to associate with complexes formed on C and D boxes of other snoRNAs, is found in association with mature U16 RNA, as well as with its precursor molecules. This fact suggests that the complex formed on the pre-mRNA remains bound to U16 throughout all the processing steps. We also show that the complex formed on the C and D boxes is necessary to stabilize mature snoRNA.

Self-cleaving motifs are found in close proximity to the sites utilized for U16 snoRNA processing.

A class of small nucleolar RNAs (snoRNAs) is encoded in introns of protein-coding genes. The U16 snoRNA belongs to this class; it is encoded in the third intron of the Xenopus laevis (Xl) L1 ribosomal protein encoding gene and is released from the pre-mRNA by processing both in vivo and in vitro systems. In this paper, we show that in close proximity to the U16 snoRNA processing sites, sequences displaying self-cleaving activity are present. These elements are conserved in the two copies of the Xl L1 and in the single copy of the X. tropicalis L1. The catalytic activity corresponds to that already described for the minimal hairpin ribozyme [Dange et al., Science 242 (1990) 585-588]; it is Mn(2+)-dependent, produces 2'-3' cyclic phosphate and 5'-OH termini and comprises an essential GAAA element. Here we show that the 2'-OH group of the G residue is essential for catalysis.

In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.

It was recently shown that a new class of small nuclear RNAs is encoded in introns of protein-coding genes and that they originate by processing of the pre-mRNA in which they are contained. Little is known about the mechanism and the factors involved in this new type of processing. The L1 ribosomal protein gene of Xenopus laevis is a well-suited system for studying this phenomenon: several different introns encode for two small nucleolar RNAs (snoRNAs; U16 and U18). In this paper, we analyzed the in vitro processing of these snoRNAs and showed that both are released from the pre-mRNA by a common mechanism: endonucleolytic cleavages convert the pre-mRNA into a precursor snoRNA with 5' and 3' trailer sequences. Subsequently, trimming converts the pre-snoRNAs into mature molecules. Oocyte and HeLa nuclear extracts are able to process X. laevis and human substrates in a similar manner, indicating that the processing of this class of snoRNAs relies on a common and evolutionarily conserved mechanism. In addition, we found that the cleavage activity is strongly enhanced in the presence of Mn2+ ions.

Two different snoRNAs are encoded in introns of amphibian and human L1 ribosomal protein genes.

We previously reported that the third intron of the X.laevis L1 ribosomal protein gene encodes for a snoRNA called U16. Here we show that four different introns of the same gene contain another previously uncharacterized snoRNA (U18) which is associated with fibrillarin in the nucleolus and which originates by processing of the pre-mRNA. The pathway of U18 RNA release from the pre-mRNA is the same as the one described for U16: primary endonucleolytic cleavages upstream and downstream of the U18 coding region produce a pre-U18 RNA which is subsequently trimmed to the mature form. Both the gene organization and processing of U18 are conserved in the corresponding genes of X.tropicalis and H.sapiens. The L1 gene thus has a composite structure, highly conserved in evolution, in which sequences coding for a ribosomal protein are intermingled with sequences coding for two different snoRNAs. The nucleolar localization of these different components suggests some common function on ribosome biosynthesis.

A novel small nucleolar RNA (U16) is encoded inside a ribosomal protein intron and originates by processing of the pre-mRNA.

We report that the third intron of the L1 ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre-mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human L1 ribosomal protein gene a highly homologous region is found which can be released from the pre-mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the L1 ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components.

The mechanisms controlling ribosomal protein L1 pre-mRNA splicing are maintained in evolution and rely on conserved intron sequences.

Sequences corresponding to the third intron of the X.laevis L1 ribosomal protein gene were isolated from the second copy of the X.laevis gene and from the single copy of X.tropicalis. Sequence comparison revealed that the three introns share an unusual sequence conservation which spans a region of 110 nucleotides. In addition, they have the same suboptimal 5' splice sites. The three introns show similar features upon oocyte microinjection: they have very low splicing efficiency and undergo the same site specific cleavages which lead to the accumulation of truncated molecules. Computer analysis and RNAse digestions have allowed to assign to the conserved region a specific secondary structure. Mutational analysis has shown that this structure is important for conferring the cleavage phenotype to these three introns. Competition experiments show that the cleavage phenotype can be prevented by coinjection of excess amounts of homologous sequences.

Identification of the sequences responsible for the splicing phenotype of the regulatory intron of the L1 ribosomal protein gene of Xenopus laevis.

Splicing of the regulated third intron of the L1 ribosomal protein gene of Xenopus laevis has been studied in vivo by oocyte microinjection of wild-type and mutant SP6 precursor RNAs and in vitro in the heterologous HeLa nuclear extract. We show that two different phenomena combine to produce the peculiar splicing phenotype of this intron. One, which can be defined constitutive, shows the same features in the two systems and leads to the accumulation of spliced mRNA, but in very small amounts. The low efficiency of splicing is due to the presence of a noncanonical 5' splice site which acts in conjunction with sequences present in the 3' portion of the intron. The second leads to the massive conversion of the pre-mRNA into site specific truncated molecules. This has the effect of decreasing the concentration of the pre-mRNA available for splicing. We show that this aberrant cleavage activity occurs only in the in vivo oocyte system and depends on the presence of an intact U1 RNA.

Inefficient in vitro splicing of the regulatory intron of the L1 ribosomal protein gene of X.laevis depends on suboptimal splice site sequences.

The splicing of the third intron of the L1 r-protein gene of X.laevis was studied in the heterologous in vitro HeLa nuclear system. Despite the evolutionary distance, the cis-elements responsible for the default process play a similar role in the two organisms. Analysis of the splicing of various mutant substrates showed that the 5' splice site is primarily responsible for the low efficiency of splicing of the third intron. The suboptimal 5' splice site sequence leads to the utilization of an upstream alternative site which corresponds to the one utilized in vivo. The accumulation of splicing intermediates in the in vitro system allowed the identification of the branch site and of the branch consensus sequence. In contrast, the in vivo regulatory mechanism involving cleavage of the pre-mRNA is not mimicked in the HeLa extract.

[Aneurysm of the splenic artery. A clinical case and review of the literature]. / Gli aneurismi dell'arteria splenica. Caso clinico e revisione della letteratura.

The Authors report a case of splenic artery aneurysm (ASA) in a 64 years old woman. Most of patients affected by ASA are asymptomatic. Rupture represents a rare complication with high mortality rate. Finally the clinical, etiopathogenetic and anatomo-pathological aspects are considered.

Immunologic conditions and infectious complications in colon and rectal surgery for neoplasms.

The authors evaluated the peroperative immunologic state of patients with colorectal tumors and controlled the postoperative incidence of infections. Twenty-one patients were studied, and delayed type hypersensitivity reactivity determined by the CMI multitest (Merieux) eight days before and eight days after surgery. A lymphocytogram was performed using monoclonal antibodies. A significant percentage of patients were anergic preoperatively. Immunologic analysis revealed lymphocytosis in the first postoperative period. The largest absolute quantitative increase was shown by NK CD16+ cells. It is possible that the results, obtained by dynamic monitoring of the main parameters of cellular immunity, will offer a new way for prognostic evaluation of surgical risk.

Endocrine, hematological and immunological changes in surgical patients undergoing general anesthesia.

Some hematological endocrine, and immunological reactions in surgical patients under general anesthesia were studied. While serum cortisol, fT4, T4, rT3 levels increased, TBG, TSH, fT3 and T3 decreased. Cortisol increase and T3 and fT3 decrease were still significant three days after surgery. TSH and T3 decrease was significantly related to cortisol increase. CD4 (helper-inducer) cells dropped, rosettes, CD 5, CD 8, CD 16 (NK) and T HLA-DR (activated) number of cells did not change significantly. The decrease in CD 4 subset was significantly related to cortisol increase. Thus surgery was followed by a reduced thyroid function and decreased CD 4 subset. However this was observed also in the few patients in whom the serum cortisol level did not change. These reactions may represent immunosuppression occurring in the postoperative period.

Gene dosage alteration of L2 ribosomal protein genes in Saccharomyces cerevisiae: effects on ribosome synthesis.

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

The accumulation of mature RNA for the Xenopus laevis ribosomal protein L1 is controlled at the level of splicing and turnover of the precursor RNA.

A specific control regulates, at the level of RNA splicing, the expression of the L1 ribosomal protein gene in Xenopus laevis. Under particular conditions, which can be summarized as an excess of free L1 protein, a precursor RNA which still contains two of the nine introns of the L1 gene accumulates. In addition to the splicing block the two intron regions undergo specific endonucleolytic cleavages which produce abortive truncated molecules. The accumulation of mature L1 RNA therefore results from the regulation of the nuclear stability of its precursor RNA. We propose that a block to splicing can permit the attack of specific intron regions by nucleases which destabilize the pre-mRNA in the nucleus. Therefore the efficiency of splicing could indirectly control the stability of the pre-mRNA.

Immunological features of endemic goiter.

Some immune aspects of simple endemic goiter have been studied through a comparison of IgG, IgA, IgM, kappa and lambda chains, and C3 and C4 in the peripheral blood of 59 patients operated on for goiter and the peripheral blood of 49 normal controls. The median IgM was lower in the goiter blood. The incidence of thyroglobulin (Tg) and microsomal (Mi) antibodies (Abs) was 20.3% in goiter blood and that of nonthyroid autoAbs was 37%. Active and total rosetted blood lymphocytes were counted and OKT3, OKT4, OKT8, Leu 1, Leu3a, Leu2b, T DR+, and NK cell populations were classified. Helper T cells were occasionally decreased when goiter was associated with lymphocytic thyroiditis. The NK percentage was sometimes higher in goiter blood, whereas the T DR+ percentage was not significantly different in the two groups. Lymphocyte infiltration (LI) was noted in 32% of goiters (about 5% with a diffuse and nodular pattern). A prevalence of helper/inducer cells was observed among the infiltrating T cells. HLA-DR antigen (Ag) positive epithelial cells were seen, not only in LI areas. Granular deposits of IgG, IgA, IgM, and C3 on the follicular basal membrane were stained in 6.7% of goiters Patterns histologically and immunologically similar to those in Hashimoto's thyroiditis may therefore be observed in long-standing simple endemic goiter, suggesting that an autoimmune mechanism may be involved in its pathogenesis.

Splicing of Xenopus laevis ribosomal protein RNAs is inhibited in vivo by antisera to ribonucleoproteins containing U1 small nuclear RNA.

The activity of antisera against ribonucleoproteins containing U1 small nuclear RNA (Sm and RNP) has been analysed on pol II transcripts in an in vivo system. Xenopus laevis ribosomal protein gene transcripts are accumulated in the form of precursor RNA when either of the two kinds of antisera are injected into the germinal vesicles of X. laevis oocytes before the injection of purified L1 and L14 ribosomal protein genes. No effect on the accumulation of mature histone mRNA is detected when X. laevis histone genes are injected together with the RNP antiserum. These results strongly suggest that U1-RNP complexes play an essential role in intron removal in vivo.

Expression of two Xenopus laevis ribosomal protein genes in injected frog oocytes. A specific splicing block interferes with the L1 RNA maturation.

The expression of two Xenopus laevis ribosomal protein genes (L1 and L14) has been analysed by microinjection of the cloned genomic sequences into frog oocyte nuclei. While the injection of the L14 gene causes the accumulation of the corresponding protein in large excess with respect to that synthesized endogenously, the L1 gene does not. Analysis of the RNA shows that both genes are actively transcribed. The seven-intron-containing L14 transcript is completely processed to a mature form, while two out of nine intron sequences persist in the L1 transcript. This precursor RNA is confined to the nucleus; its accumulation is due to a specific block of splicing operating at the level of two defined introns and not to saturation of the processing apparatus of the oocyte. The different behaviour of the two genes may reflect different mechanisms of regulation which, in the case of the L1 gene, could operate at the level of splicing.

Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.