Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order. (C) 2012 Elsevier Ltd. All rights reserved.

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LTG33D), originally produced by some enterotoxigenic Escherichia coil (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LIG33D. Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LTG33D elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LTG33D. Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LTG33D. Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine. (C) 2011 Elsevier Ltd. All rights reserved.

Chemical characterization and in vitro biological activity of four tropical legumes, Styzolobium aterrimum L., Styzolobium deeringianum, Leucaena leucocephala, and Mimosa caesalpiniaefolia, as compared with a tropical grass, Cynodon spp. for the use in ruminant diets

Leucaena leucocephala (LEU) and three under-utilized tanniferous legumes, Styzolobium aterrimum L. (STA), Styzolobium deeringianum (STD), and Mimosa caesalpiniaefolia Benth (MIC) were chemically characterized and the biological activity of tannins was evaluated using in vitro simulated ruminal fermentation through tannin-binding polyethylene glycol (PEG) and compared with a non-tanniferous tropical grass hay, Cynodon spp. (CYN). The Hohenheim gas test was used and gas production (GP) was recorded at 4, 8, 12, 24, 32, 48, 56, 72, 80, and 96 h incubation with and without PEG. Kinetic parameters were estimated by an exponential model. STA, STD, and LEU contained higher (P < 0.05) crude protein than MIC, which had greater neutral detergent fibre and acid detergent fibre. Total phenols, total tannins, and condensed tannins (CT) were consistently the highest in MIC. Gas production was the lowest from MIC (P < 0.05) and the highest in LEU and STA. MIC + PEG largely reduced (P < 0.05) the lag phase and the fractional rate of fermentation and increased potential GP. Kinetic parameters of STA + PEG and LEU + PEG were not affected. LEU + PEG produced greater gas increment (P < 0.05) than STD + PEG, although both legumes had the same CT. All legumes except MIC were more extensively degraded than CYN. However, fermentation of the legumes was differently affected by the presence and proportions of CT, indigestible fibre or both.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes

The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

Haplotypes of susceptibility to chronic periodontitis in the Interleukin 8 gene do not influence protein level in the gingival crevicular fluid

Objective: Previously, we identified that the ATC/TTC haplotype formed by polymorphisms in the Interleukin-(IL)8 gene conferred susceptibility to chronic periodontitis (CP). The aim of the study was to investigate whether the IL8 haplotype ATC/TTC was associated with the volume of gingival crevicular fluid (GCF), the concentration of interleukin IL-8 in the GCF, as well as periodontal conditions in patients with CP in comparison to controls without CP. Methods: Seventy-nine individuals (CP: n = 41, controls: n = 38) were grouped according to the presence (susceptible for CP) or absence (not susceptible for CP) of the IL8 ATC/TTC haplotype. After periodontal clinical evaluation, they were subdivided by the presence or absence of CP. GCF was collected from each patient and the IL-8 levels were determined by ELISA. The GCF volume of each subject was measured by means of a calibrated electronic device. Comparisons of means between carriers and non-carriers of the ATC/TTC haplotype were evaluated using the Mann-Whitney test. Linear regression and stepwise linear regression analysis were used to analyse the association of the GCF volume with potential covariates and their contribution for the phenotype. Results: We did not find significant differences of both periodontal conditions and IL-8 concentration in the GCF of patients with the presence or absence of the IL8 ATC/TTC haplotype. However, the GCF volume was significantly higher amongst the patients affected by CP that are absent for the IL8 ATC/TTC haplotype. In addition, linear regression analysis showed a statistically significant association between GCF volume and CP, IL8 haplotype ATC/TTC and IL-8 concentration. Conclusions: The IL8 haplotype of susceptibility to CP was neither associated with IL-8 cytokine levels nor with clinical periodontal parameters. Also, CP, IL8 haplotype and IL-8 concentration showed a positive association with the GCF volume levels in the studied patients. (c) 2012 Published by Elsevier Ltd.

Nitrogen excretion during embryonic development of the green iguana, Iguana iguana (Reptilia; Squamata)

Development within the cleidoic egg of birds and reptiles presents the embryo with the problem of accumulation of wastes from nitrogen metabolism. Ammonia derived from protein catabolism is converted into the less toxic product urea or relatively insoluble uric acid. The pattern of nitrogen excretion of the green iguana, Iguana iguana, was determined during embryonic development using samples from allantoic fluid and from the whole homogenized egg, and in hatchlings and adults using samples of blood plasma. Urea was the major excretory product over the course of embryonic development. It was found in higher concentrations in the allantoic sac, suggesting that there is a mechanism present on the allantoic membrane enabling the concentration of urea. The newly hatched iguana still produced urea while adults produced uric acid. The time course of this shift in the type of nitrogen waste was not determined but the change is likely to be related to the water relations associated with the terrestrial habit of the adult. The green iguana produces parchment-shelled eggs that double in mass during incubation due to water absorption: the eggs also accumulate 0.02 mM of urea, representing 82% of the total measured nitrogenous residues that accumulate inside the allantois. The increase in egg mass and urea concentration became significant after 55 days of incubation then were unchanged until hatching. (C) 2012 Elsevier Inc. All rights reserved.

Cholesterol-lowering effect of whole lupin (Lupinus albus) seed and its protein isolate

This study describes the hypocholesterolaemic effect of whole lupin and its protein in hamsters. The diets were: casein (control group HC), lupin protein isolate (group HPI) and whole lupin seed (group HWS). Diets from HPI and HWS promoted a significant reduction of total cholesterol and non-HDL cholesterol in the hamsters' plasma as compared with HC. The true digestibility of HPI and HC groups were similar and differed significantly from the HWS one, which in turn showed a significant difference in total sterol excretion as compared to the former groups. Histological analysis of the liver revealed that animals fed on HPI and HWS diets presented a low level of steatosis (level 1) as compared to the ones fed on HC diet (level 4). Our findings demonstrate that protein isolate from Lupinus albus from Brazil has a metabolic effect on endogenous cholesterol metabolism and a protector effect on development of hepatic steatosis. (C) 2011 Elsevier Ltd. All rights reserved.

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na, K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (gamma C-33) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K+, ATP and N-4(+)center dot K-0.5 for Na+ was unaffected. Exogenous pig FXYD2 increased the V-max for stimulation of gill Na,K-ATPase activity by Na+, K+ and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na, K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity. (C) 2012 Elsevier B.V. All rights reserved.

Association of AXIN2 with Non-syndromic Oral Clefts in Multiple Populations

We have previously shown the association of AXIN2 with oral clefts in a US population. Here, we expanded our study to explore the association of 11 AXIN2 markers in 682 cleft families from multiple populations. Alleles for each AXIN2 marker were tested for transmission distortion with clefts by means of the Family-based Association Test. We observed an association with SNP rs7224837 and all clefts in the combined populations (p = 0.001), and with SNP rs3923086 and cleft lip and palate in Asian populations (p = 0.004). We confirmed our association findings in an additional 528 cleft families from the United States (p < 0.009). We tested for gene-gene interaction between AXIN2 and additional cleft susceptibility loci. We assessed and detected Axin2 mRNA and protein expression during murine palatogenesis. In addition, we also observed co-localization of Axin2 with Irf6 proteins, particularly in the epithelium. Our results continue to support a role for AXIN2 in the etiology of human clefting. Additional studies should be performed to improve our understanding of the biological mechanisms linking AXIN2 to oral clefts.

The bactericidal effect of human secreted group IID phospholipase A(2) results from both hydrolytic and non-hydrolytic activities

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca2+-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10 mu g/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca2+-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca2+-independent damaging activity may play a role in the bactericidal function of the protein. (C) 2012 Elsevier Masson SAS. All rights reserved.

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

Abstract Background The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. Results We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. Conclusion The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

Procalcitonin (PCT) and C-reactive Protein (CRP) as severe systemic infection markers in febrile neutropenic adults

Abstract Background Procalcitonin (PCT) is an inflammatory marker that has been used as indicator of severe bacterial infection. We evaluated the concentrations of PCT as a marker for systemic infection compared to C-reactive protein (CRP) in patients neutropenic febrile. Methods 52 adult patients were enrolled in the study. Blood sample was collected in order to determine the serum concentrations of PCT, CRP and other hematological parameters at the onset of fever. The patients were divided into 2 groups, one with severe infection (n = 26) and the other in which the patients did not present such an infection (n = 26). Then PCT and CRP concentrations at the fever onset were compared between groups using non parametric statistical tests, ROC curve, sensitivity, specificity, likelihood ratio, and Spearman's correlation coefficient. Results The mean of PCT was significantly higher in the group with severe infection (6.7 ng/mL versus 0.6 ng/mL – p = 0.0075) comparing with CRP. Serum concentrations of 0.245 ng/mL of PCT displayed 100% de sensitivity and 69.2% specificity. PCT concentrations of 2,145 ng/mL presented a likelihood ratio of 13, which was not observed for any concentration of CRP. Conclusion PCT seems to be an useful marker for the diagnosis of systemic infection in febrile neutropenic patients, probably better than CRP.

Androgen responsive intronic non-coding RNAs

Abstract Background Transcription of large numbers of non-coding RNAs originating from intronic regions of human genes has been recently reported, but mechanisms governing their biosynthesis and biological functions are largely unknown. In this work, we evaluated the existence of a common mechanism of transcription regulation shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line. Results Using a custom-built cDNA microarray enriched in intronic transcribed sequences, we found 39 intronic non-coding RNAs for which levels were significantly regulated by androgen exposure. Orientation-specific reverse transcription-PCR indicated that 10 of the 13 were transcribed in the antisense direction. These transcripts are long (0.5–5 kb), unspliced and apparently do not code for proteins. Interestingly, we found that the relative levels of androgen-regulated intronic transcripts could be correlated with the levels of the corresponding protein-coding gene (asGAS6 and asDNAJC3) or with the alternative usage of exons (asKDELR2 and asITGA6) in the corresponding protein-coding transcripts. Binding of the androgen receptor to a putative regulatory region upstream from asMYO5A, an androgen-regulated antisense intronic transcript, was confirmed by chromatin immunoprecipitation. Conclusion Altogether, these results indicate that at least a fraction of naturally transcribed intronic non-coding RNAs may be regulated by common physiological signals such as hormones, and further corroborate the notion that the intronic complement of the transcriptome play functional roles in the human gene-expression program.

Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in virological sustained responder and non-responder patients

Abstract Background About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. Findings Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. Conclusions Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site.