Pilot study on circulating miRNA signature in children with obesity born small for gestational age and appropriate for gestational age.

BACKGROUND: Children born small for gestational age (SGA) are at increased risk of metabolic dysfunction. Dysregulation of specific microRNAs (miRNAs) contributes to aberrant gene expression patterns underlying metabolic dysfunction. OBJECTIVE: We aimed to determine and compare circulating miRNA (c-miRNA) profile of SGA and appropriate for gestational age (AGA) children with obesity and with normal weight, in order to identify biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity. METHODS: Small non-coding RNAs from serum of 15 SGA children with obesity (OB-SGA), 10 SGA children with normal weight (NW-SGA), 17 AGA children with obesity (OB-AGA) and 12 AGA children with normal weight (NW-AGA) (mean age 11.2 ± 2.6) have been extracted and sequenced in order to detect and quantify miRNA expression profiles. RESULTS: RNA-seq analyses showed 28 miRNAs dysregulated in OB-SGA vs. NW-SGA and 19 miRNAs dysregulated in OB-AGA vs. NW-AGA. Among these, miR-92a-3p, miR-122-5p, miR-423-5p, miR-484, miR-486-3p and miR-532-5p were up regulated, and miR-181b-5p was down regulated in both OB-SGA and OB-AGA compared with normal weight counterparts. Pathway analysis and miRNA target prediction suggested that these miRNAs were particularly involved in insulin signalling, glucose transport, insulin resistance, cholesterol and lipid metabolism. CONCLUSION: We identified a specific profile of c-miRNAs in SGA and AGA children with obesity compared with SGA and AGA children with normal weight. These c-miRNAs could represent specific biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity.

Regulation of IGFBP3 gene expression in short children born small for gestational age.

OBJECTIVE: Approximately 6% of newborns at term are small for gestational age (SGA) and present a birth weight and/or length less than -2SD from the mean. SGA infants are at increased risk for perinatal morbidity, associated psychological and/or mental problems, persistent short stature (about 15% of subjects) and metabolic alterations. Insulin-like growth factors (IGFs), their common receptor (IGF1R) and their binding proteins (IGFBPs) play a critical role in fetal and postnatal growth. In these genes common polymorphisms, such as single nucleotide polymorphisms and variable number of tandem repeats, have been investigated with conflicting results with respect to SGA-related outcomes, and the functional role of these gene variants remains to be elucidated. DESIGN: The study group consisted of 100 pre-pubertal short children born SGA and 94 healthy controls, matched for sex and age, recruited at the Department of Biomedicine of Development Age of the Bari University and at the Paediatric Department of the Messina Hospital. In the present study we analyzed the allelic frequency of the polymorphisms -795 G/A, -667 G/A, -396 C/T in the IGFBP3 in SGA children and their influence on the basal and insulin-stimulated transcriptional activity of the gene. RESULTS: We found that the polymorphisms -667 G/A and -396 C/T in the IGFBP3 promoter region are capable of having an effect on the transcriptional activity of the gene, although with opposing effects. Interestingly, the -667 G/A polymorphism has a negative impact on the IGFBP3 transcription, while the -396 C/T polymorphism determines an increase of the transcriptional activity of the IGFBP3 gene promoter. Interestingly, we found that the -396 C/T polymorphism correlates with lower birth length in SGA children. Most importantly, while the diminished IGFBP3 transcriptional activity induced by the -667A polymorphism was significantly recovered after insulin administration (p-value<0.05), the increased transcriptional activity caused by the -396T polymorphism was not restored to baseline levels by insulin. CONCLUSIONS: Altogether our results demonstrated that the -667 G/A and the -396 C/T polymorphisms in IGFBP3 promoter region influence the basal transcriptional activity of the gene.

Results of resection for hilar cholangiocarcinoma with analysis of prognostic factors.

BACKGROUND/AIMS: Resection for hilar cholangiocarcinoma remains a challenging procedure and recent published results continue to show that few patients are cured of their disease. The objective of this review was to determine the results of radical resection and to identify factors associated with long-term survival. METHODOLOGY: Retrospective review of resection for hilar cholangiocarcinoma in 29 consecutive patients with statistical analysis of prognostic factors, including p53 status. RESULTS: The mortality and morbidity rates were 6.9% and 34%, respectively. The overall 5-year survival was 20% with the median survival being 16 months. Univariate analysis identified the following factors associated with poor survival; involved lymph nodes, vascular invasion, advanced tumor stage, positive tumor margins, and p53 mutation. However, none of these factors was associated with poor survival by multivariate analyses. CONCLUSIONS: Radical resection for hilar cholangiocarcinoma can be performed with acceptable morbidity and mortality, but most patients succumb to their disease. p53 status may be a helpful adjunct to routine pathological staging.

New p53 mutations in hilar cholangiocarcinoma.

BACKGROUND: Inactivation of the tumour suppressor gene, p53, is the commonest genetic abnormality in human cancer. The study of the type of p53 mutation in a given tumour may provide prognostic information, clues to aetiology and become useful for therapeutics. MATERIALS AND METHODS: The molecular characterisation of p53 was performed by restriction analysis, denaturing gradient gel electrophoresis, and gene sequencing for exons 5-9. RESULTS: We report, p53 mutational analysis in exons 5-9 in 29 European patients with hilar cholangiocarcinoma who underwent attempted resection. Four patients (14%) showed somatic single nucleotide substitutions with amino acid changes (146, 163, 175, 158, and 175) with one showing mutations in two different positions involving a loss of two CfoI sites. All the mutations occurred in exon 5. Three patients had a germline polymorphism (10%) with a silent substitution in codon 213 (exon 6). CONCLUSIONS: The systematic screening for p53 mutations in European patients with hilar cholangiocarcinoma has shown that the type of mutation (except 175) is different and its incidence is much lower when compared to the pattern previously reported for intrahepatic cholangiocarcinoma in East Asian patients. A probable explanation is that the presence and type of p53 mutation is dependent on geographic and environmental factors which vary in different populations.

Mutation of p53 Tumor Suppressor Gene in Hepatocellular Carcinoma.

In recent years, the most commonly observed genetic alteration in hepatocellular carcinoma (HCC), as in many other tumors affecting man, has been reported to be the mutation of the p53 coding gene (1,2). This gene has the features of a recessive oncosuppressor in its wild-type form and can be a dominant oncogene in its mutated form. The gene (20 kb) is located in a single copy on the short arm of chromosome 17 and contains 11 exons interrupted by 10 introns. The mRNA (2.8 kb) codes for a protein of 393 amino acids, which is expressed at relatively low levels in all tissues. p53 product is a 53-kDa phosphoprotein involved in the regulation of cell cycle, in DNA synthesis and repair, and in cell differentiation and apoptosis (see refs. 3-6, for reviews).

Characterization of p53 mutations in colorectal liver metastases and correlation with clinical parameters.

The presence and type of mutations of the p53 tumor suppressor gene were determined in 40 patients undergoing curative hepatic resection for metastatic colorectal carcinoma. This represents the largest series in the literature on the screening of p53 mutations for liver metastases. The analysis was performed in exons 5-9 by denaturing gradient gel electrophoresis followed by direct sequencing. Forty-five percent of tumors showed mutation in p53, and this was observed only in exons 5-8. Mutations at codon positions 167, 196, 204, 213, 245, 281, 282, 286, and 306; deletion of codon 251 and of the first nucleotide of codon 252; and Leu residue (CTC) insertion downstream codon 252 are reported for the first time in colorectal liver metastasis. Mutations at codon positions 163, 248, and 273 have been reported previously. Correlation of p53 status with clinical parameters showed that patients with mutated p53 had a statistically higher number of lesions when compared with patients with wild-type p53 (P<0.050). In particular, of patients with mutated p53, 41% had three or more metastases compared with 14% of patients with wild-type p53. Synchronous metastases were present in 70% of the patients with p53 mutations and in only 29% of patients with wild-type p53 (P<0.025). In addition, patients with p53 mutations are more likely to develop recurrence (73%) compared with patients with wild-type p53 (33%; P<0.001). Other factors considered, including preoperative carcinoembryonic antigen level, bilobar distribution, and size of the lesion(s), did not show significant correlation with p53 status. These results suggest that p53 status might be an important prognostic indicator to predict the pattern and likelihood of treatment failure after hepatic resection.

Guinea pig p53 mRNA: identification of new elements in coding and untranslated regions and their functional and evolutionary implications.

We report the sequence of the guinea pig p53 cDNA. The comparative analysis of the coding and noncoding regions of p53 cDNAs of all available complete vertebrate sequences has allowed us to single out new conserved signals possibly involved in p53 functional activity. We have focused our attention on the most variable region of the protein, the proline (P)-rich domain, suggested to play a fundamental role in antiproliferative pathways. In this domain we have identified the PXXXXP repeated motif and singled out a common consensus sequence that can be considered a signature for mammalian p53: PXXXXPX{0,4}PX{0,9}PA(T,P,I,)(S,P)WPL. We have demonstrated the significance of the PXXXXP motif in SH3-binding protein and suggested its structure to be a loop. Also, the 5' and 3' untranslated regions (UTRs) of the guinea pig were sequenced, and this study represents the first detailed structural analysis of the UTRs of the p53 mRNAs available in literature. The 5' UTR of guinea pig (233 nt) can be folded into a stable secondary structure resembling that predicted in mouse. The 3' UTR of guinea pig is 771 nt long and shows higher similarity with human than with rodent sequences, having a region of about 350 nt that is deleted in rat and mouse. In the 3' UTR we have identified the presence of a mammalian-wide interspersed repeat sequence and of a cytoplasmic polyadenylation element, which could be involved in translational activation by promoting polyadenylation of mRNA, providing information about a possible mechanism of regulation of p53 expression mediated by the 3' UTR of the mRNA. The observations presented here could open new avenues to targeted mutations and experimental approaches useful in investigating new regulation mechanisms of p53 translation, activity, and stability.

Update of AMmtDB: a database of multi-aligned metazoa mitochondrial DNA sequences.

The present paper describes AMmtDB, a database collecting the multi-aligned sequences of vertebrate mitochondrial genes coding for proteins and tRNAs, as well as the multiple alignment of the mammalian mtDNA main regulatory region (D-loop) sequences. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. As far as the genes coding for tRNAs are concerned, the multi-alignments based on the primary and the secondary structures are both provided; for the mammalian D-loop multi-alignments we report the conserved regions of the entire D-loop (CSB1, CSB2, CSB3, the central region, ETAS1 and ETAS2) as defined by Sbisà et al. [ Gene (1997), 205, 125-140). A flatfile format for AMmtDB has been designed allowing its implementation in SRS (http://bio-www.ba.cnr.it:8000/BioWWW/#AMMTDB ). Data selected through SRS can be managed using GeneDoc or other programs for the management of multi-aligned data depending on the user's operative system. The multiple alignments have been produced with CLUSTALV and PILEUP programs and then carefully optimized manually.

Folding and binding activity of the 3'UTRs of Paracentrotus lividus bep messengers.

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3'UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lengths of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3'UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.

p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation.

Forty-two patients with hepatocellular carcinoma (HCC) were resected and their tumours were analysed for p53 mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) and gene sequencing. All the exons have been analysed in this study. Eight of 12 HCCs with cirrhosis due to viral hepatitis and the two patients with sarcomatoid changes displayed p53 mutations. In contrast, no mutation was observed in the fibrolamellar variant (n = 9), non-cirrhotics (n = 13) and alcoholic cirrhosis (n = 6). The mutations observed were in exons 5-8. Two mutations were observed in codons 136 and 213 as well as a T insertion between residues 156 and 157 (exon 5) and these are reported for the first time in HCC. Likewise, the silent mutation polymorphism in codon 213 was noticed in 3 of the 42 patients. Survival analysis of these patients after surgery showed the mean and median survival in patients with wild-type p53 to be 60 and 43 months respectively. In the group with p53 mutations, the mean and median survival was 15 and 12 months. The difference was statistically significant (P= 0.003).

A 54-kDa protein specifically associates the 3' untranslated region of three maternal mRNAs with the cytoskeleton of the animal part of the Paracertrotus lividus egg.

Bep mRNAs, i.e., maternal messengers coding for cell surface proteins, are localized in the animal part of Paracertrotus lividus egg and embryos. Here we have examined the involvement of the cytoskeleton in asymmetric distribution of bep3 mRNA. Moreover, in order to understand whether and how cis- and trans-acting factors are necessary for bep3 mRNA localization, we have looked for in vitro-specific interactions between egg proteins and bep3 mRNA. By northwestern assay we have identified a 54-kDa protein that binds to the 3'UTR of bep3 mRNA. This 54-kDa protein also permits association of 3'UTR of bep3 with cytoskeleton elements, indicating its involvement in the localization process. Binding of 54-kDa protein to 3'UTR of bep1 and bep4 has also been demonstrated, suggesting that a binding motif is shared with these other two mRNAs of the same gene family. Northwestern analyses carried out utilizing proteins extracted from different developmental stages indicate that the 54-kDa protein is the only protein able to bind to the 3'UTR of bep3.

Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications.

This paper reports the first comprehensive analysis of Displacement loop (D-loop) region sequences from ten different mammalian orders. It represents a systematic evolutionary study at the molecular level on regulatory homologous regions in organisms belonging to a well defined class, mammalia, which radiated about 150 million years ago (Mya). We have aligned and analyzed 26 complete D-loop region sequences available in the literature and the fat dormouse sequence, recently determined in our laboratory. The novelty of our alignment consists of the extensive manual revision of the preliminary output obtained by computer program to optimize sequence similarity, particularly for the two peripheral domains displaying heterogeneity in length and the presence of repeated sequences. The multialignment is available at the WWW site: http://www.ba.cnr.it/dloop.html. Our comparative study has allowed us to identify new conserved sequence blocks present in all the species under consideration and events of insertion/deletion which have important implications in both functional and evolutionary aspects. In particular we have detected two blocks, about 60 bp long, extended termination associated sequences (ETAS1 and ETAS2) conserved in all the organisms considered. Evaluation against experimental work suggests a possible functional role of ETAS1 and ETAS2 in the regulation of replication and transcription and targeted experimental approaches. The analyses on conserved sequence blocks (CSBs) clearly indicate that CSB1 is the only very essential element, common to all mammalian mt genomes, while CSB2 and CSB3 could be involved in different though related functions, probably species specific, and thus more linked to nuclear mitochondrial coevolutionary processes. Our hypothesis on the different functional implications of the conserved elements, CSBs and TASs, reported so far as main regulatory signals, would explain the different conservation of these elements in evolution. Moreover the intra-order comparison of the D-loop regions highlights peculiar features useful to define the evolutionary dynamics of this region in closely related species.

The evolution of the RNase P- and RNase MRP-associated RNAs: phylogenetic analysis and nucleotide substitution rate.

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments.

Human mitochondrial tRNA processing.

tRNA processing is a central event in mammalian mitochondrial gene expression. We have identified key enzymatic activities (ribonuclease P, precursor tRNA 3'-endonuclease, and ATP(CTP)-tRNA-specific nucleotidyltransferase) that are involved in HeLa cell mitochondrial tRNA maturation. Different mitochondrial tRNA precursors are cleaved precisely at the tRNA 5'- and 3'-ends in a homologous mitochondrial in vitro processing system. The cleavage at the 5'-end precedes that at the 3'-end, and the tRNAs are substrates for the specific CCA addition in the same in vitro system. Using a comparative enzymatic approach as well as biochemical and immunological techniques, we furthermore demonstrate that human cells contain two distinct enzymes that remove 5'-extensions from tRNA precursors, the previously characterized nuclear and the newly identified mitochondrial ribonuclease P. These two cellular isoenzymes have different substrate specificities that seem to be well adapted to their structurally disparate mitochondrial and nuclear tRNA substrates. This kind of approach may also help to understand the structural diversities and commonalities of tRNAs.

RNase mitochondrial RNA processing cleaves RNA from the rat mitochondrial displacement loop at the origin of heavy-strand DNA replication.

Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non-coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site-specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement-loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in-vivo transcriptional analysis of the rat displacement-loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition.

Transcription of rat mitochondrial NADH-dehydrogenase subunits. Presence of antisense and precursor RNA species.

We have characterized the transcriptional pattern of the rat mitochondrial ND6-containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable than the L transcripts. We have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNA(Glu). The mapping of the ND1 5' end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature.

Detection of novel transcripts in the human mitochondrial DNA region coding for ATPase8-ATPase6 subunits.

We have analyzed the tRNA(Lys), ATPase8, ATPase6, COIII region of mitochondrial DNA in several human tissues. Beside the mature tRNA(Lys), ATPase8 and ATPase6 common mRNA, and COIII mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNA(Lys) plus the ATPase8 and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.

The evolution of the mitochondrial D-loop region and the origin of modern man.

The origin of modern man is a highly debated issue that has recently been tackled by using mitochondrial DNA sequences. The limited genetic variability of human mtDNA has been explained in terms of a recent common genetic ancestry, thus implying that all modern-population mtDNAs originated from a single woman who lived in Africa less than 0.2 Mya. This divergence time is based on both the estimation of the rate of mtDNA change and its calibration date. Because different estimates of the rate of mtDNA evolution can completely change the scenario of the origin of modern man, we have reanalyzed the available mitochondrial sequence data by using an improved version of the statistical model, the "Markov clock," devised in our laboratory. Our analysis supports the African origin of modern man, but we found that the ancestral female from which all extant human mtDNAs originated lived in a time span of 0.3-0.8 Mya. Pushing back the date of the deepest root of the human implies that the earliest divergence would have been in the Homo erectus population.

Transcription mapping of the Ori L region reveals novel precursors of mature RNA species and antisense RNAs in rat mitochondrial genome.

We have identified new transcripts in the region surrounding the L-strand replication origin (Ori L) of rat liver mitochondrial DNA. In particular, we have detected previously unidentified intermediates of RNA processing on both the heavy and the light strands, such as precursors of the ND2 mRNA plus the Trp-tRNA and precursors of the tRNAs clustered in the Ori L region. This indicates that the mechanism of RNA processing in mitochondria proceeds step-wise producing a variety of precursors of the mature forms. The other striking finding is the detection of antisense RNA species in the region of L-strand replication. Since a variety of antisense transcripts were also found in the D-loop region of rat mitochondrial DNA, we suggest that they might play a regulatory role in the replication and expression of the mitochondrial genome.

The branching order of mammals: phylogenetic trees inferred from nuclear and mitochondrial molecular data.

In order to clarify some controversial phylogenies such as those regarding the triplet of human, rodent, and cow and the evolutionary position of Lagomorpha with respect to other mammals, we have analyzed both nuclear and mitochondrial genes using the stationary Markov model developed in our laboratory. We found that the two sets of genes give different results. In particular the mitochondrial tree showed rabbit linked first to rodents and the rabbit-rodents branch linked to artiodactyls with human as the outgroup. The most favorite nuclear tree showed human linked first to artiodactyls and the human-artiodactyls branch linked to rabbit with rodents as the outgroup. The obvious questions, (1) which tree is the correct one, or (2) both trees can be incorrect, and (3) how can we explain such an evolutionary pattern, are discussed on the basis of our limited knowledge of factors that influence the clocklike behavior of biological macromolecules.