Small bowel adenocarcinoma in a patient with a 5-year history of untreated Crohn's disease: a case report.

AIM: Complication of Crohn's disease (CD) of the small intestine is small bowel adenocarcinoma (SBA). A lot of studies on Crohn's disease have estimated the increased relative risk of small bowel carcinoma compared to the general population. In clinical settings, it is difficult to detect SBA in CD, therefore most of cases are diagnosed after surgery for strictures without suspicion of malignancy. CASE REPORT: The present case concerns a 48-year-old man with a suspicious 5-year history of untreated chronic inflammatory bowel disease. The patient was admitted to our unit with persistent abdominal pain, 20 kg weight loss and intestinal obstruction, confirmed at CT scans. It was performed an emergency laparotomy, terminal ileus was resected and intestinal continuity was restored. Histological examination revealed a poorly differentiated adenocarcinoma. DISCUSSION: The risk factors of SBA include long-standing and extensive Crohn's disease, young age, male sex, smoke, early onset, complications such as strictures and fistulas The most common clinical presentation of small bowel carcinoma in Crohn's disease is intestinal obstruction accompanied by wheight loss. The diagnosis is very difficult because imaging techniques may not be able to differentiate areas of small bowel carcinomas from benign fibrotic or acute inflammatory strictures. CONCLUSION: Small bowel adenocarcinoma is a rare disease but this evenience must be considered in patients with strictures for Crohn's disease. Preoperative diagnosis is still highly challenging despite significant radiological and endoscopic progress.

Expression of vitellogenin receptor gene in the ovary of wild and captive Atlantic bluefin tuna (Thunnus thynnus).

The cDNA sequences of vitellogenin receptor proteins (VgR(+) and VgR(-)), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR(-) gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR(-). The total length of VgR(+) cDNA was 4006 nucleotides (nt) and that of VgR(-) was 3946 nt. Relative amounts of VgR(-) were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR(-) were less in individuals with yolked oocytes (ripening stage, May-June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR(-) is not under oestrogen control. During the ripening period, greater VgR(-) gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna.

Comparative study of liver vitellogenin gene expression and oocyte yolk accumulation in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.).

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fish's migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels of VgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.

Diagnosis and Molecular Variability of an Argentinean Population of Nacobbus aberrans with Some Observations on Histopathology in Tomato.

Diagnosis of an Argentinean population of Nacobbus sp. infecting sweet pepper (lamuyo) was carried out including morphology, scanning electron microscopy, and molecular studies. In light of our morphometric, molecular and host-range results, we consider the studied population to belong to N. aberrans s. l., and by host range tests the population is assigned to the "sugar beet group." ITS-PCR analysis on individual male and immature female specimens of this population yielded amplification products of approximately 922 bp. RFLP profiles and sequencing of the ITS region revealed that, in addition to the host group, the present population can be assigned to the "Argentina 2" group. Disease development and histopathology were investigated with glasshouse observations using tomato, pepper, sugar beet and potato seedlings exposed to nematode infection for 45 days at 28 +/- 2 degrees C. Histopathology of tomato roots confirmed that all immature stages and young females and males are migratory, whereas mature females are obligate sedentary endoparasites. Rather than syncytia, large regions of cortical necrosis and cavities were detected in tomato swellings infected by juveniles. However, syncytia were associated only with adult females. Large root galls, hyperplasia, abnormal proliferation of lateral roots and asymmetry of root structure were common anatomical changes induced by the nematode feeding in tomato roots.

Characterization and Sequence Variation in the rDNA Region of Six Nematode Species of the Genus Longidorus (Nematoda).

Total DNA was isolated from individual nematodes of the species Longidorus helveticus, L. macrosoma, L. arthensis, L. profundorum, L. elongatus, and L. raskii collected in Switzerland. The ITS region and D1-D2 expansion segments of the 26S rDNA were amplified and cloned. The sequences obtained were aligned in order to investigate sequence diversity and to infer the phylogenetic relationships among the six Longidorus species. D1-D2 sequences were more conserved than the ITS sequences that varied widely in primary structure and length, and no consensus was observed. Phylogenetic analyses using the neighbor-joining, maximum parsimony and maximum likelihood methods were performed with three different sequence data sets: ITS1-ITS2, 5.8S-D1-D2, and combining ITS1-ITS2+5.8S-D1-D2 sequences. All multiple alignments yielded similar basic trees supporting the existence of the six species established using morphological characters. These sequence data also provided evidence that the different regions of the rDNA are characterized by different evolution rates and by different factors associated with the generation of extreme size variation.

Horizontal transfer of a bacterial gene involved in polyglutamate biosynthesis to the plant-parasitic nematode Meloidogyne artiellia.

Analysis of a genomic fragment from the plant parasitic nematode Meloidogyne artiellia revealed the presence of a gene which, in bacteria, is involved in the formation of polyglutamate capsule. Searching of various databases, including the Caenorhabditis elegans genome sequence and the large EST datasets from a variety of parasitic nematodes, showed that no similar genes have been identified in other nematodes or in any other eukaryotic organisms. The M. artiellia gene has a typical eukaryotic structure and its mRNA is present in the intestine. The gene is expressed in all life cycle stages tested. These findings demonstrate horizontal gene transfer may be important in catalyzing the diversification of nematode lineages.

Nematode chitin synthases: gene structure, expression and function in Caenorhabditis elegans and the plant parasitic nematode Meloidogyne artiellia.

Although the presence of chitin in nematodes is well documented little is known about its synthesis in this phyletic group. The recently completed genome sequence of Caenorhabditis elegans predicts two sequences with homology to chitin synthases (chitin-UDP acetyl-glucosaminyl transferase; EC 2.4.1.16). We show that these genes are differentially expressed in a pattern that may reflect different functional roles. One gene is expressed predominantly in the adult hermaphrodite (the main egg-producing stage in the nematode) and later larval stages, which is consistent with a role in production of chitin for the eggshell. The other gene, however, is expressed in the cells that form the pharynx, and only in the period directly preceding a moult. These data suggest that the product of this gene is involved in synthesis of the feeding apparatus, which is replaced during each moult. We have also isolated a full-length genomic sequence of a chitin synthase orthologue from the plant parasitic nematode Meloidogyne artiellia. The single gene present in M. artiellia shows an expression pattern that is consistent with a role for the protein in production of the eggshell.

Evolutionary genomics in Metazoa: the mitochondrial DNA as a model system.

One of the most important aspects of mitochondrial (mt) genome evolution in Metazoa is constancy of size and gene content of mtDNA, whose plasticity is maintained through a great variety of gene rearrangements probably mediated by tRNA genes. The trend of mtDNA to maintain the same genetic structure within a phylum (e.g., Chordata) is generally accepted, although more recent reports show that a considerable number of transpositions are observed also between closely related organisms. Base composition of mtDNA is extremely variable. Genome GC content is often low and, when it increases, the two complementary bases distribute asymmetrically, creating, particularly in vertebrates, a negative GC-skew. In mammals, we have found coding strand base composition and average degree of gene conservation to be related to the asymmetric replication mechanism of mtDNA. A quantitative measurement of mtDNA evolutionary rate has revealed that each of the various components has a different evolutionary rate. Non-synonymous rates are gene specific and fall in a range comparable to that of nuclear genes, whereas synonymous rates are about 22-fold higher in mt than in nuclear genes. tRNA genes are among the most conserved but, when compared to their nuclear counterparts, they evolve 100 times faster. Finally, we describe some molecular phylogenetic reconstructions which have produced unexpected outcomes, and might change our vision of the classification of living organisms.

Lineage-specific evolution of echinoderm mitochondrial ATP synthase subunit 8.

Peculiar evolutionary properties of the subunit 8 of mitochondrial ATP synthase (ATPase8) are revealed by comparative analyses carried out between both closely and distantly related species of echinoderms. The analysis of nucleotide substitution in the three echinoids demonstrated a relaxation of amino acid functional constraints. The deduced protein sequences display a well conserved domain at the N-terminus, while the central part is very variable. At the C-terminus, the broad distribution of positively charged amino acids, which is typical of other organisms, is not conserved in the two different echinoderm classes of the sea urchins and of the sea stars. Instead, a motif of three amino acids, so far not described elsewhere, is conserved in sea urchins and is found to be very similar to the motif present in the sea stars. Our results indicate that the N-terminal region seems to follow the same evolutionary pattern in different organisms, while the maintenance of the C-terminal part in a phylum-specific manner may reflect the co-evolution of mitochondrial and nuclear genes.

Modulation of expression at the level of splicing of cut-1 RNA in the infective second-stage juvenile of the plant parasitic nematode Meloidogyne artiellia.

The cut-1 gene coding for cuticlin-1 has been isolated from the plant parasitic nematode Meloidogyne artiellia. The sequence of the cut-1 gene was compared with the corresponding sequence from the free-living nematode Caenorhabditis elegans. The high degree of similarity between the amino acid sequences, together with the occurrence of characteristic sequence motifs, indicates that the cuticlin-1 is a non-collagenous component of the cuticle also in plant parasitic nematodes. Studies on the expression pattern during the development of M. artiellia indicate that there is a burst of expression of this gene during moulting. Then, the expression rate is reduced in the infective juveniles, which migrate in the soil. In the sedentary females, in contrast, no expression is detected, while in the males which move freely through the soil, the gene is expressed and the transcript fully processed. These data strongly suggest that the gene is developmentally regulated. It is proposed that the production of cuticlin plays an important role in determining the mechanical properties of the cuticle. Furthermore, evidence is provided to indicate that the modulation of cut-1 expression is achieved by regulation of the cis-splicing mechanism in the infective second-stage juvenile.

A silent trans-splicing signal in the cuticlin-encoding gene of the plant-parasitic nematode Meloidogyne artiellia.

A gene (cut-1) coding for a cuticular protein, cuticlin-1, has been isolated and sequenced in the plant parasitic nematode, Meloidogyne artiellia. The nucleotide sequence revealed a typical eukaryotic-like organization, exons and introns bordered by canonical sequences. The 5' flanking region presents nematode-specific sequence motifis, including a trans-splicing signal. Studies on the expression of this gene demonstrated that, while in the adult females cut-1 is not expressed, the removal of the introns occurs in the eggs. These experiments also indicate that cis-splicing precedes the processing of the 5' untranslated region. In no case has a trans-spliced transcript been detected.

Complete sequence of the mitochondrial DNA in the sea urchin Arbacia lixula: conserved features of the echinoid mitochondrial genome.

The complete nucleotide sequence (15,719 nucleotides) of the mitochondrial DNA (mtDNA) from the sea urchin Arbacia lixula is presented. The comparison of gene arrangement between different echinoderm orders of the same class provides evidence that the gene organization is conserved within the same echinoderm class. The peculiarities of sea urchin mtDNA features, already described, are confirmed by the A. lixula mtDNA sequence. The comparison of the entire sequences of mtDNA among A. lixula, Paracentrotus lividus, and Strongylocentrotus purpuratus allowed us to detect peculiar features, common to the three sea urchin species, that can represent the molecular signature of the mt genome in the sea urchin group. Analysis of the nucleotide composition indicates that A. lixula mtDNA, in contrast with the mtDNA of other sea urchins, shows a bias in the use of T and tends to avoid the use of C, most evident in the neutral part of the molecule, such as the third codon positions. This observation indicates that the three sea urchin mtDNAs evolve under different mutation pressure. Analysis of the sequence evolution allowed us to confirm the phylogenetic tree. However, the absolute divergence time, calculated on the basis of paleontological estimates, largely diverged from the expected one.

Formalin-induced infidelity in PCR-amplified DNA fragments.

A 643-nucleotide-long fragment of rDNA gene was amplified by PCR in the nematode worm Caenorhabditis elegans. When the experiments were performed by using samples fixed in formalin, artefacts were detected. While the size of the amplified fragment resulted unaffected, very striking differences were seen in the nucleotide sequences of the amplified fragments. Furthermore, in many cases, the PCR reaction failed completely. The results obtained might warn of potential problems, especially when the amount of DNA to be amplified is scarce.

Cytochrome oxidase subunit III from Arbacia lixula: detection of functional constraints by comparison with homologous sequences.

In this paper we report the comparison of the sequences of the cytochrome oxidase subunit III from three different sea urchin species. Both nucleotide and amino acid sequences have been analyzed. The nucleotide sequence analysis reveals that the sea urchin sequences obey some rules already found in mammals. The base substitution analysis carried out on the sequences of the three species pairs, shows that the evolutionary dynamics of the first and the second codon positions are so slow that do not allow a quantitative measurement of their genetic distances, thus demonstrating that also in these species the COIII gene is strongly conserved during evolution. Changes occurring at the third codon positions indicate that the three species evolved from a common ancestor under different directional mutational pressure. The multi-alignment of the sea urchin proteins indicates the existence of the amino acid sequence motif N R T that represents a possible glycosylation site. Another glycosylation site has been detected in the mammalian cytochrome oxidase subunit III, in a position slightly different. Such an analysis revealed, for the first time, a new functional aspect of this sequence.

Mitochondrial DNA detection and copy number determination in the spermatozoa of the sea urchin Arbacia lixula.

The Polymerase Chain reaction technique has been used in order to detect and amplify a specific region of mtDNA, in a total DNA preparation extracted from the sperm of the sea urchin Arbacia lixula. The amplified fragment is the D-loop region which hybridizes with the homologous region extracted from the egg mtDNA. The results demonstrate that mtDNA is present in sperm cell, and, since the replication origin is present it is potentially able to replicate in the zygote. Furthermore, the technique used allowed us to estimate mtDNA copy number in sea urchin sperm, which has never been done before. Our results are that sea urchin sperm cell contains between 4 and 28 mtDNA molecules.

Mitochondrial DNA in the sea urchin Arbacia lixula: evolutionary inferences from nucleotide sequence analysis.

From the stirodont Arbacia lixula we determined the sequence of 5,127 nucleotides of mitochondrial DNA (mtDNA) encompassing 18 tRNAs, two complete coding genes, parts of three other coding genes, and part of the 12S ribosomal RNA (rRNA). The sequence confirms that the organization of mtDNA is conserved within echinoids. Furthermore, it underlines the following peculiar features of sea urchin mtDNA: the clustering of tRNAs, the short noncoding regulatory sequence, and the separation by the ND1 and ND2 genes of the two rRNA genes. Comparison with the orthologous sequences from the camarodont species Paracentrotus lividus and Strongylocentrotus purpuratus revealed that (1) echinoids have an extra piece on the amino terminus of the ND5 gene that is probably the remnant of an old leucine tRNA gene; (2) third-position codon nucleotide usage has diverged between A. lixula and the camarodont species to a significant extent, implying different directional mutational pressures; and (3) the stirodont-camarodont divergence occurred twice as long ago as did the P. lividus-S. purpuratus divergence.

Mitochondrial DNA in the sea urchin Arbacia lixula: nucleotide sequence differences between two polymorphic molecules indicate asymmetry of mutations.

Two polymorphic forms of mitochondrial DNA (mtDNA) extracted from Arbacia lixula eggs were cloned and the nucleotide sequences of specific regions determined. A comparison of the sequences of the sense strand of the two molecules demonstrates that all the differences are transitions and only of the A----G type. A change such as G----A (or A----G) on the sense mtDNA strand results from either a direct G----A (or A----G) mutation on that strand or a C----T (or T----C) on the complementary strand. None of the C----T (or T----C) changes were detected on the sense strand, which implies that the A----G mutation bias on the sense strand is not reversed for the other strand. Our observation indicates the existence of mechanisms acting asymmetrically on the two mtDNA strands, possibly during mtDNA replication.

Direct evidence that restriction endonucleases may under estimate the degree of divergence between molecules.

We studied two polymorphic forms of mtDNA extracted from A. lixula eggs. In order to compare and to quantitate the variability, we sequenced specific regions of the two molecules. In this way, we obtained a precise measurement of the variability within two haplotypes. We also obtained a direct demonstration that some differences in nucleotide sequence can escape detection when restriction endonuclease analysis is used. Our results underline the unreliability of the use of restriction mapping to estimate divergence between relatively short and closely related DNA sequences.

Mitochondrial genome in animal cells. Structure, organization, and evolution.

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are presented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.