Global DNA promoter methylation in frontal cortex of alcoholics and controls

To determine if ethanol consumption and alcoholism cause global DNA methylation disturbances, we examined alcoholics and controls using methylation specific microarrays to detect all annotated gene and non-coding microRNA promoters and their CpG islands. DNA was isolated and immunoprecipitated from the frontal cortex of 10 alcoholics and 10 age and gender-matched controls then labeled prior to co-hybridization. A modified Kolmogorov–Smirnov test was used to predict differentially enriched regions (peaks) from log-ratio estimates of amplified vs input DNA. More than 180,000 targets were identified for each subject which correlated with > 30,000 distinct, integrated peaks or high probability methylation loci. Peaks were mapped to regions near 17,810 separate annotated genes per subject representing hypothetical methylation targets. No global methylation differences were observed between the two subject groups with 80% genetic overlap, but extreme methylation was observed in both groups at specific loci corresponding with known methylated genes (e.g., H19) and potentially other genes of unknown methylation status. Methylation density patterns targeting CpG islands visually correlated with recognized chromosome banding. Our study provides insight into global epigenetic regulation in the human brain in relationship to controls and potentially novel targets for hypothesis generation and follow-up studies of alcoholism.     

Telomere biology in Metazoa

In this review we present critical overview of some of the available literature on the fundamental biology of telomeres and telomerase in Metazoan. With the exception of Nematodes and Arthropods, the (TTAGGG)_n sequence is conserved in most Metazoa. Available data show that telomerase-based end maintenance is a very ancient mechanism in unicellular and multicellular organisms. In invertebrates, fish, amphibian, and reptiles persistent telomerase activity in somatic tissues might allow the maintenance of the extensive regenerative potentials of these species. Telomerase repression among birds and many mammals suggests that, as humans, they may use replicative aging as a tumor protection mechanism.     

Ancestral modal Y-STR haplotype shared among Romani and South Indian populations

One of the primary unanswered questions regarding the dispersal of Romani populations concerns the geographical region and/or the Indian caste/tribe that gave rise to the proto-Romani group. To shed light on this matter, 161 Y-chromosomes from Roma, residing in two different provinces of Serbia, were analyzed. Our results indicate that the paternal gene pool of both groups is shaped by several strata, the most prominent of which, H1-M52, comprises almost half of each collection's patrilineages. The high frequency of M52 chromosomes in the two Roma populations examined may suggest that they descend from a single founder that has its origins in the Indian subcontinent. Moreover, when the Y-STR profiles of haplogroup H derived individuals in our Roma populations were compared to those typed in the South Indian emigrants from Malaysia and groups from Madras, Karnataka (Lingayat and Vokkaliga castes) and tribal Soligas, sharing of the two most common haplotypes was observed. These similarities suggest that South India may have been one of the contributors to the proto-Romanis. European genetic signatures (i.e., haplogroups E1b1b1a1b-V13, G2a-P15, I-M258, J2-M172 and R1-M173), on the other hand, were also detected in both groups, but at varying frequencies. The divergent European genetic signals in each collection are likely the result of differential gene flow and/or admixture with the European host populations but may also be attributed to dissimilar endogamous practices following the initial founder effect. Our data also support the notion that a number of haplogroups including G2a-P15, J2a3b-M67(xM92), I-M258 and E1b1b1-M35 were incorporated into the proto-Romani paternal lineages as migrants moved from northern India through Southwestern Asia, the Middle East and/or Anatolia into the Balkans.     

Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using Real Time RT-PCR and immunohistochemistry

Purpose

Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes.

Methods

Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2^− ΔΔCt method.

Results

23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%.

Conclusion

Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique.     

Investigation of the association between ATP2B4 and ATP5B genes with colorectal cancer

Colorectal cancer (CRC) develops as a multi-step process which results from gradual accumulation of mutations in proto-oncogenes, tumor suppressor, and DNA repair genes. Mortality rate of CRC is very high. Therefore, development of alternative diagnostic methods which can be used in the early diagnosis is crucial. ATP2B4 gene encodes one of the four isoforms of p-type ATPase PMCA enzyme and bears critical importance in maintaining the balance of intracellular calcium homeostasis by providing the export of calcium ions out of the cell. ATP5B encodes a subunit of the mitochondrial ATP synthase which is an f-type ATPase. In this study, the relationship between ATP2B4 and ATP5B genes and CRC regarding gene expression was investigated. Study groups were constructed from a number of 50 patients (25 males, 25 females) with the mean age of 55.68 ± 9.4 and the gene expression levels in the healthy and cancerous tissues of the patients were compared by using semi-quantitative PCR and Real-Time PCR methods. As a result, in patients with rectum tumors, there was a significant relationship between ATP2B4 gene expression and the tumor location and in patients younger than 45 years, ATP5B gene expressions were detected significantly higher in tumor tissues by using RT-PCR. However, no significant relationship was detected in terms of expression differences of ATP2B4 and ATP5B genes between cancerous and healthy tissues of the CRC patients. ATP2B4 and ATP5B genes might have indirect associations in CRC pathogenesis and the investigation of their interactions with DNA repair and other related genes may help in understanding of CRC formation.     

Cloning, localization and differential expression of the Trypanosoma cruzi TcOGNT-2 glycosyl transferase

It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPβ and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle.     

Gene regulatory network from microarray data of colon cancer patients using TSK-type recurrent neural fuzzy network

In this work we applied a TSK-type recurrent neural fuzzy approach to extract regulatory relationship among genes and reconstruct gene regulatory network from microarray data. The identified signature has captured the regulatory relationship among 27 differentially expressed genes from microarray dataset. We applied three different methods viz., feed forward neural fuzzy, modified genetic algorithm and recurrent neural fuzzy, on the same data set for the inference of GRNs and the results obtained are almost comparable. In all tested cases, TRNFN identified more biologically meaningful relations. We found that 87.8% of the total interactions extracted by TRNFN are correct in accordance with the biological knowledge. Our analysis resulted in 2 major outcomes. First, upregulated genes are regulated by more genes than downregulated genes. Second, tumor activators activate other tumor activators and suppress tumor suppressers strongly in the disease environment. These findings will help to elucidate the common molecular mechanism of colon cancer, and provide new insights into cancer diagnostics, prognostics and therapy.     

Dynamic covariation between gene expression and genome characteristics

Gene and protein expression is controlled so that cells can react to changing intra- and extracellular signals by modulating biochemical networks and pathways. We have previously shown that gene expression and the properties of expressed proteins are dynamically correlated. Here we investigated correlations between gene related parameters and gene expression patterns, and found statistically significant correlations in microarray datasets for different cell types, organisms and processes, including human B and T cell stimulation, cell cycle in HeLa cells, infection in intestinal epithelial cells, Drosophila melanogaster life span, and Saccharomyces cerevisiae cell cycle. Our method was applied to time course datasets individually for each time point. We derived from sequence information numerous parameters for nucleotide composition, two-base composition, codon usage, skew parameters, and codon bias. In addition to coding regions, we also investigated correlations for complete genes and introns. Significant dynamic correlations were identified for each of the analyses. Our method also proved useful for detecting dynamic shifts in gene expression profiles, such as in the D. melanogaster dataset. Detection of changes in the properties of expressed genes and proteins might be useful for predicting or following biological processes, responses, growth, differentiation and possibly in related disorders.     

The complete chloroplast genome of Origanum vulgare L. (Lamiaceae)

Oregano (Origanum vulgare L., Lamiaceae) is a medicinal and aromatic plant maybe best known for flavouring pizza. New applications e.g. as natural antioxidants for food are emerging due to the plants' high antibacterial and antioxidant activity. The complete chloroplast (cp) genome of Origanum vulgare (GenBank/EBML/DDBJ accession number: JX880022) consists of 151,935 bp and includes a pair of inverted repeats (IR) of 25,527 bp separated by one small and one large single copy region (SSC and LSC) of 17,745 and 83,136 bp, respectively.     

Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype ‘TAS-R8’. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production.     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus)

A proliferation-inducing ligand (APRIL) is an important member of the tumor necrosis factor (TNF) superfamily. In the present study, a novel cDNA was isolated from the spleen of goat by RT-PCR and designated as goat APRIL (gAPRIL). The open reading frame (ORF) of this cDNA covered 753 bp, encoding a protein of 250 amino acids. Sequence comparison showed that gAPRIL contains a predicted transmembrane domain, a putative furin protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF gene in mammals. The predicted three dimensional (3D) structure of soluble part of the gAPRIL (gsAPRIL) monomer analyzed by comparative protein modeling revealed that it is very similar to its counterparts. Real-time PCR analysis revealed that gAPRIL was constitutively expressed in various tissues. Recombinant gsAPRIL fused with NusA tag was efficiently produced in Escherichia coli BL21 (DE3) and then analyzed by the SDS-PAGE as well as western blot. Laser scanning confocal microscopy analysis showed gsAPRIL could bind to its receptors. In vitro, the MTT and flow cytometric methods revealed that purified gsAPRIL protein was not only able to promote survival/proliferation of goat splenocytes, but also able to stimulate survival/proliferation of mouse B cells. These results indicated that gAPRIL plays an important role in survival/proliferation of goat splenocytes and provided a basis for investigating its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and as an immunotherapeutic in goats.     

Investigating the effects of the presence of foreign DNA on DNA methylation and DNA repair events in cultured eukaryotic cells

Methylation of DNA in eukaryotic cells, global as well as gene-specific, is affected by endogenous and endogenous factors. In this paper, it is reported that deviations in DNA methylation and expression of genes involved in DNA repair and the cell cycle are affected in 143B cultured cells containing an expression vector. Global DNA methylation analysis with cytosine-extension assay revealed a decreased global DNA methylation in the presence of the expression vector. Less promoter-specific methylation, as measured by bisulfite-MS PCR, was observed for MGMT and p16INK4a in vector-containing cells. Comet assay investigations revealed a negative effect on the DNA repair capacity of both BER and NER in Complex III compromised cells. This was reflected in the down-regulation of hOGG1 and ERCC1 expression. The results presented in this paper support the existence of a strong relationship between impaired mitochondrial function and deviations in DNA methylation and extend this relationship to impaired DNA repair.     

Silencing efficiency differs among tissues and endogenous microRNA pathway is preserved in short hairpin RNA transgenic mice

In short hairpin RNA (shRNA) transgenic mice, the tissue difference in gene silencing efficiency and oversaturation of microRNA (miRNA) pathway have not been well assessed. We studied these problems in our previously-reported anti-copper/zinc superoxide dismutase (SOD1) shRNA transgenic mice. Although there was a tissue difference (liver and skeletal muscle, >95%; central nervous system and lung, ∼80%), the target gene silencing was systemic and our anti-SOD1 shRNA transgenic mice recapitulated the SOD1-null mice. Neither endogenous miRNAs nor their target gene levels were altered, indicating the preservation of endogenous miRNA pathways. We think that the shRNA transgenic mice can be utilized for gene analysis.