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1.  Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter 
Gene  2013;536(1):135-144.
Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The proximal E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide −300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.
PMCID: PMC3926136  PMID: 24315822
EPHX1; Sp1; Sp3; Transcriptional regulation; Alternative Gene Promoters; Biotransformation
2.  Increased expression of mdig predicts poorer survival of the breast cancer patients 
Gene  2013;535(2):218-224.
Breast cancer is the most common cancer and the second leading cause of cancer death among women of all races and Hispanic origin populations in the United States. In the present study, we reported that the survival time of the breast cancer patients is influenced by the expression level of mdig, a previously identified lung cancer-associated oncogene encoding a JmjC-domain protein. By checking the expression levels of mRNA and protein of mdig through both RT-PCR and immunohistochemistry in samples from 204 patients, we noticed that about 30% of breast cancer samples showed increased expression of mdig. Correlation of the mdig expression levels with the survival time of the breast cancer patients indicated a clear inverse relationship between mdig expression and patient survival, including poorer overall survival, distant metastasis free survival, relapse free survival, and post-progression survival. Taken together, these data suggest that an increased expression of mdig is an important prognostic factor for poorer survival time of the breast cancer patients.
PMCID: PMC3976998  PMID: 24309373
Breast cancer; Mdig; Prognosis; Patient survival
3.  Molecular cloning, characterisation and mRNA expression of the ryanodine receptor from the peach-potato aphid, Myzus persicae 
Gene  2015;556(2):106-112.
The peach potato aphid, Myzus persicae, is one of the most important agricultural pests of temperate climates. It is mainly controlled through the judicious application of insecticides; however, over time, aphids have developed resistance to many insecticidal classes. The recent introduction of synthetic diamide insecticides, with a novel mode of action, potentially offers new tools to control aphid populations. These diamides act on the ryanodine receptor (RyR), a large endoplasmic calcium release channel. In this study we have cloned cDNAs encoding the complete open reading frame of the RyR from M. persicae. The open reading frame is 15,306 base pairs long and encodes a protein of 5101 amino acids. The aphid RyR shares many of the features of other insect and vertebrate RyRs, including a highly conserved transmembrane region. However, unlike the other RyRs characterised to date, the M. persicae channel does not display alternative splicing at any stage of its developmental cycle, so it cannot generate functional variants of the channel.
•We obtained the full-length cDNA sequence of the M. persicae RyR.•The ORF is 15,306 bp long and encodes a protein of 5101 amino acids.•The aphid RyR shares many common features with other insect and vertebrate RyRs.•The aphid channel does not display alternative splicing at any developmental stage.•Aphids cannot generate variants of the RyR channel, unlike other insects.
PMCID: PMC4309888  PMID: 25447916
Ryanodine receptor; Insect; Diamide insecticides; Alternative splicing; Aphid
4.  Construction of shuttle vectors for genetic manipulation and molecular analysis of Mycobacteria1 
Gene  1997;190(1):37-44.
Two novel shuttle vectors for mycobacteria are described which have been derived from the expression system pSD5 developed in our laboratory. Plasmid pSD5B is a promoter-selection vector containing a promoterless lacZ gene and allows the identification of mycobacterial promoters by the blue colour of the colonies on solid media containing XGal. Moreover, the chronological order of appearance of blue colonies and intensity of colour provide a qualitative index of transcriptional strengths of the cloned promoters. Plasmid pSD5C has been designed to construct mycobacterial genomic libraries and express the cloned DNA inserts as fusion proteins with maltose binding protein in mycobacteria. Libraries in pSD5C provide feasibility for their screening with either DNA probes or specific antisera for identifying the genes of interest and for isolation of specific genetic loci by complementation of Escherichia coli and mycobacterial mutants. These vectors combine the ease of working in E. coli with the advantage of directly propagating them in mycobacteria without further manipulations. Finally, we demonstrate that these vectors function efficiently both in fast growing Mycobacterium smegmatis and slow growing mycobacteria including Mycobacterium tuberculosis and Mycobacterium bovis BCG.
PMCID: PMC4320809  PMID: 9185847
Gene expression; Promoter; Genomic library; Fusion protein; Genetic complementation
5.  The Role of DNA Methylation in Regulation of the Murine Lhx3 Gene 
Gene  2013;534(2):10.1016/j.gene.2013.10.045.
LHX3 is a LIM-homeodomain transcription factor with critical roles in pituitary and nervous system development. Mutations in the LHX3 gene are associated with pediatric diseases featuring severe hormone deficiencies, hearing loss, developmental delay, and other symptoms. The mechanisms that govern LHX3/Lhx3 transcription are poorly understood. In this study, we examined the role of DNA methylation in the expression status of the mouse Lhx3 gene. Pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with 5-aza-2’-deoxycytidine, a demethylating reagent. This treatment lead to activation of Lhx3 gene expression suggesting that methylation contributes to Lhx3 regulation. Treatment of Pit-1/0 pituitary cells with a combination of a demethylating reagent and a histone deacetylase inhibitor led to rapid activation of Lhx3 expression, suggesting possible crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA was subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation at specific sites in the Lhx3 locus compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the MeCp2 methyl binding protein and Lhx3 promoter regions in the Pit-1/0 cell line. Overall, this study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.
PMCID: PMC3870101  PMID: 24183897
transcription; promoter; chromatin; LHX3
6.  Extraordinarily low evolutionary rates of short wavelength-sensitive opsin pseudogenes 
Gene  2013;534(1):10.1016/j.gene.2013.09.114.
Aquatic organisms such as cichlids, coelacanths, seals, and cetaceans are active in UV-blue color environments, but many of them mysteriously lost their abilities to detect these colors. The loss of these functions is a consequence of the pseudogenization of their short wavelength-sensitive (SWS1) opsin genes without gene duplication. We show that the SWS1 gene (BdenS1ψ) of the deep-sea fish, pearleye (Benthalbella dentata), became a pseudogene in a similar fashion about 130 million years ago (Mya) yet it is still transcribed. The rates of nucleotide substitution (~1.4 × 10−9 /site/year) of the pseudogenes of these aquatic species as well as some prosimian and bat species are much smaller than the previous estimates for the globin and immunoglobulin pseudogenes.
PMCID: PMC3852691  PMID: 24125953
Aquatic animals; SWS1 pseudogenes; Molecular evolution
7.  Genetic variants in Selenoprotein P plasma 1 gene (SEPP1) are associated with fasting insulin and first phase insulin response in Hispanics 
Gene  2013;534(1):10.1016/j.gene.2013.10.035.
Insulin resistance is not fully explained on a molecular level, though several genes and proteins have been tied to this defect. Knockdowns of the SEPP1 gene, which encodes the Selenoprotein P (SeP) protein, have been shown to increase insulin sensitivity in mice. SeP is a liver-derived plasma protein and a major supplier of selenium, which is a proposed insulin mimetic and antidiabetic agent.
SEPP1 single nucleotide polymorphisms (SNPs) were selected for analysis with glucometabolic measures.
Participants and Measures
1424 Hispanics from families in the Insulin Resistance Atherosclerosis Family Study (IRASFS). Additionally, the multi-ethnic Insulin Resistance Atherosclerosis Study was used. A frequently sampled intravenous glucose tolerance test was used to obtain precise measures of acute insulin response (AIR) and the insulin sensitivity index (SI).
21 SEPP1 SNPs (tagging SNPs (n=12) from HapMap, 4 coding variants and 6 SNPs in the promoter region) were genotyped and analyzed for association.
Two highly correlated (r2=1) SNPs showed association with AIR (rs28919926; Cys368Arg; p=0.0028 and rs146125471; Ile293Met; p=0.0026) while rs16872779 (intronic) was associated with fasting insulin levels (p=0.0097). In the smaller IRAS Hispanic cohort, few of the associations seen in the IRASFS were replicated, but meta-analysis of IRASFS and all 3 IRAS cohorts (N= 2446) supported association of rs28919926 and rs146125471 with AIR (p=0.013 and 0.0047, respectively) as well as rs7579 with SI (p=0.047).
Overall, these results in a human sample are consistent with the literature suggesting a role for SEPP1 in insulin resistance.
PMCID: PMC3856675  PMID: 24161883
Acute Insulin Response (AIR); Selenium; Selenoproteins; Insulin Resistance; Fibrinogen; Hispanic Americans
8.  Sexual differences of imprinted genes' expression levels 
Gene  2013;533(1):10.1016/j.gene.2013.10.006.
In mammals, genomic imprinting has evolved as a dosage-controlling mechanism for a subset of genes that play critical roles in their unusual reproduction scheme involving viviparity and placentation. As such, many imprinted genes are highly expressed in sex-specific reproductive organs. In the current study, we sought to test whether imprinted genes are differentially expressed between the two sexes. According to the results, the expression levels of the following genes differ between the two sexes of mice: Peg3, Zim1, Igf2, H19 and Zac1. The expression levels of these imprinted genes are usually greater in males than in females. This bias is most obvious in the developing brains of 14.5-dpc embryos, but also detected in the brains of postnatal-stage mice. However, this sexual bias is not obvious in 10.5-dpc embryos, a developmental stage before the sexual differentiation. Thus, the sexual bias observed in the imprinted genes is most likely attributable by gonadal hormones rather than by sex chromosome complement. Overall, the results indicate that several imprinted genes are sexually different in terms of their expression levels, and further suggest that the transcriptional regulation of these imprinted genes may be influenced by unknown mechanisms associated with sexual differentiation.
PMCID: PMC3872986  PMID: 24125951
Genomic imprinting; Sexual dimorphism; Peg3; Igf2; Zac1
9.  Molecular cloning and characterization of SL3: A stem cell-specific SL RNA from the planarian Schmidtea mediterranea 
Gene  2013;533(1):156-167.
Spliced leader (SL) trans-splicing is a biological phenomenon, common among many metazoan taxa, consisting in the transfer of a short leader sequence from a small SL RNA to the 5′ end of a subset of pre-mRNAs. While knowledge of the biochemical mechanisms driving this process has accumulated over the years, the functional consequences of such post-transcriptional event at the organismal level remain unclear. In addition, the fact that functional analyses have been undertaken mainly in trypanosomes and nematodes leaves a somehow fragmented picture of the possible biological significance and evolution of SL trans-splicing in eukaryotes. Here, we analyzed the spatial expression of SL RNAs in the planarian flatworm Schmidtea mediterranea, with the goal of identifying novel developmental paradigms for the study of trans-splicing in metazoans. Besides the previously identified SL1 and SL2, S. mediterranea expresses a third SL RNA described here as SL3. While, SL1 and SL2 are collectively expressed in a broad range of planarian cell types, SL3 is highly enriched in a subset of the planarian stem cells engaged in regenerative responses. Our findings provide new opportunities to study how trans-splicing may regulate the phenotype of a cell.
PMCID: PMC3994195  PMID: 24120894
Stem cells; SL RNA; Planarians; Regeneration; Trans-splicing
10.  Genetic complexity of the human surfactant-associated proteins SP-A1 and SP-A2 
Gene  2012;531(2):126-132.
Pulmonary surfactant protein A (SP-A) plays a key role in innate lung host defense, in surfactant-related functions, and in parturition. In the course of evolution, the genetic complexity of SP-A has increased, particularly in the regulatory regions (i.e. promoter, untranslated regions). Although most species have a single SP-A gene, two genes encode SP-A in humans and primates (SFTPA1and SFTPA2). This may account for the multiple functions attributed to human SP-A, as well as the regulatory complexity of its expression by a relatively diverse set of protein and non-protein cellular factors. The interplay between enhancer cis-acting DNA sequences and trans-acting proteins that recognize these DNA elements is essential for gene regulation, primarily at the transcription initiation level. Furthermore, regulation at the mRNA level is essential to ensure proper physiological levels of SP-A under different conditions. To date, numerous studies have shown significant complexity of the regulation of SP-A expression at different levels, including transcription, splicing, mRNA decay, and translation. A number of trans-acting factors have also been described to play a role in the control of SP-A expression. The aim of this report is to describe the genetic complexity of the SFTPA1 and SFTPA2 genes, as well as to review regulatory mechanisms that control SP-A expression in humans and other animal species.
PMCID: PMC3570704  PMID: 23069847
surfactant proteins; innate immunity; genetic variants; transcriptional regulation; evolution
11.  Kinesin-5: cross-bridging mechanism to targeted clinical therapy 
Gene  2013;531(2):133-149.
Kinesin motor proteins comprise an ATPase superfamily that goes hand in hand with microtubules in every eukaryote. The mitotic kinesins, by virtue of their potential therapeutic role in cancerous cells, have been a major focus of research for the past 28 years since the discovery of the canonical Kinesin-1 heavy chain. Perhaps the simplest player in mitotic spindle assembly, Kinesin-5 (also known as Kif11, Eg5, or kinesin spindle protein, KSP) is a plus-end-directed motor localized to interpolar spindle microtubules and to the spindle poles. Comprised of a homotetramer complex, its function primarily is to slide anti-parallel microtubules apart from one another. Based on a multi-faceted analysis of this motor from numerous laboratories over the years, we have learned a great deal about the function of this motor at the atomic level for catalysis and as an integrated element of the cytoskeleton. These data have, in turn, informed the function of motile kinesins on the whole, as well as spearheaded integrative models of the mitotic apparatus in particular and regulation of the microtubule cytoskeleton in general. We review what is known about how this nanomotor works, its place inside the cytoskeleton of cells, and its small-molecule inhibitors that provide a toolbox for understanding motor function and for anticancer treatment in the clinic.
PMCID: PMC3801170  PMID: 23954229
kinesin; motor protein; phylogeny; evolution; cytoskeletal motor; catalysis; ATP hydrolysis; mitosis; mitotic spindle; microtubule; mitotic motor protein; targeted inhibitor; Eg5; KSP; Kif11; structural biology; ispinesib; monastrol; allosteric inhibition; L5 loop; review
12.  Gudu, an Armadillo repeat-containing protein, is required for spermatogenesis in Drosophila 
Gene  2013;531(2):10.1016/j.gene.2013.08.080.
The Drosophila annotated gene CG5155 encodes a protein that contains 10 Armadillo-repeats and has an unknown function. To fill this gap, we performed loss-of-function studies using RNAi. By analysis of four independent Drosophila RNAi lines targeting two non-overlapping regions of the CG5155 transcript, we demonstrate that this gene is required for male fertility. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with the bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 is a subfamily of Armadillo-repeat containing proteins that may have an evolutionarily conserved function in spermatogenesis.
PMCID: PMC3839114  PMID: 24055424
Gudu; Drosophila; Spermatogenesis; Individualization complex; ARMC4; Armadillo repeats
13.  Structure and function of the DNA ligases encoded by the mammalian LIG3 gene 
Gene  2013;531(2):10.1016/j.gene.2013.08.061.
Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.
PMCID: PMC3881560  PMID: 24013086
Gene  2013;531(1):10.1016/j.gene.2013.08.021.
Cystathionine β-synthase (CBS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)), found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype.
PMCID: PMC3824618  PMID: 23981774
Homocystinuria; cystathionine beta-synthase; immunocytochemistry; protein misfolding
15.  Genetic variation of the cutaneous HPA axis: An analysis of UVB-induced differential responses 
Gene  2013;530(1):10.1016/j.gene.2013.08.035.
Mammalian skin incorporates a local equivalent of the hypothalamic-pituitary-adrenal (HPA) axis that is critical in coordinating homeostatic responses against external noxious stimuli. Ultraviolet radiation B (UVB) is a skin-specific stressor that can activate this cutaneous HPA axis. Since C57BL/6 (B6) and DBA/2J (D2) strains of mice have different predispositions to sensorineural pathway activation, we quantified expression of HPA axis components at the gene and protein levels in skin incubated ex vivo after UVB or sham irradiation. Urocortin mRNA was up-regulated after all doses of UVB with a maximum level at 50 mJ/cm2 after 12 h for D2 and at 200 mJ/cm2 after 24 h for B6. Proopiomelanocortin mRNA was enhanced after 6 h with the peak after 12 h and at 200 mJ/cm2 for both genotypes of mice. ACTH levels in tissue and media increased after 24 h in B6 but not in D2. UVB stimulated β-endorphin expression was higher in D2 than B6. Melanocortin receptor 2 mRNA was stimulated by UVB in a dose-dependent manner, with a peak at 200 mJ/cm2 after 12 h for both strains. The expression of Cyp11a1 mRNA — a key mitochondrial P450 enzyme in steroidogenesis, was stimulated at all doses of UVB irradiation, with the most pronounced effect after 12–24 h. UVB radiation caused, independently of genotype, a dose-dependent increases in corticosterone production in the skin, mainly after 24 h of histoculture. Thus, basal and UVB stimulated expression of the cutaneous HPA axis differs as a function of genotype: D2 responds to UVB earlier and with higher amplitude than B6, while B6 shows prolonged (up to 48 h) stress response to a noxious stimulus such as UVB.
PMCID: PMC3807248  PMID: 23962689
ultraviolet B radiation; HPA axis; cutaneous stress; C57Bl/6; DBA2J
16.  Polyadenylation Site-specific Differences in the Activity of the Neuronal βCstF-64 Protein in PC-12 Cells 
Gene  2013;529(2):220-227.
Recent genome-wide analyses have implicated alternative polyadenylation — the process of regulated mRNA 3′ end formation — as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, βCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that βCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for βCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that βCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that βCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the β-adducin mRNA. Notably, we demonstrate that the activity of βCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the βCstF-64 protein. Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.
PMCID: PMC3783208  PMID: 23948079
alternative polyadenylation; alternative splicing; neuronal gene expression; CstF-64; β-adducin
17.  Splicing variants of the porcine betaine-homocysteine S-methyltransferase gene: implications for mammalian metabolism 
Gene  2013;529(2):228-237.
Betaine-homocysteine S-methyltransferase (BHMT) activity is only detected in the liver of rodents, but in both the liver and kidney cortex of humans and pigs; therefore, the pig was chosen as a model to define the spatial and temporal expression of BHMT during development. During fetal development, a total of ten splice variants of bhmt were expressed at varying levels across a wide range of porcine tissues. Two variants contained an identical ORF that encoded a C-terminal truncated form of BHMT (tBHMT). The bhmt transcripts were expressed at significant levels in the liver and kidney from day 45 of gestation (G45) onward. The transcripts encoding tBHMT represented 5–13% of the total bhmt transcripts in G30 fetus, G45 liver, and adult liver and kidney cortex. The dominant structural feature of wild type BHMT is an (βα)8 barrel, however, a modeled structure of tBHMT suggests this protein would assume a horseshoe fold and lack methyltransferase activity. Low BHMT activity was detected in the G30 fetus, and slightly increased levels of activity were observed in the liver from G45 and G90 fetuses. The bhmt promoter contained three key CpG sites, and methylation of these sites was significantly higher in adult lung compared to adult liver. The data reported herein suggest that genomic DNA methylation and variation of the 5' and 3' UTRs of bhmt transcripts are key regulators for the level of BHMT transcription and translation.
PMCID: PMC3894628  PMID: 23948084
betaine; homocysteine; splice variant; bisulfite sequencing
18.  Resistin disrupts glycogen synthesis under high insulin and high glucose levels by down-regulating the hepatic levels of GSK3β 
Gene  2013;529(1):50-56.
The effect of mouse resistin on hepatic insulin resistance in vivo and in vitro, and its possible molecular mechanism were examined. Focusing on liver glycogen metabolism and gluconeogenesis, which are important parts of glucose metabolism, in primary cultures of rat hepatocytes we found that glycogen content was significantly lower (P<0.05) after treatment with recombinant murine resistin only in the presence of insulin plus glucose stimulation. Protein levels of factors in the insulin signaling pathway involved in glycogen synthesis were examined by Western blot analysis, with the only significant change observed being the level of phosphorylated (at Ser 9) glycogen synthase kinase-3β (GSK-3β) (P<0.001). No differences in the protein levels for the insulin receptor β (IRβ), insulin receptor substrates (IRS1 and IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) or their phosphorylated forms were observed between control and resistin treated primary rat hepatocytes. In a mouse model with high liver-specific expression of resistin, fasting blood glucose levels and liver glycogen content changed. Fasting blood glucose levels were significantly higher (P<0.001) in the model mice, compared to the control mice, while the glycogen content of the liver tissue was about 60% of that of the control mice (P<0.05). The gluconeogenic response was not altered between the experimental and control mice. The level of phosphorylated GSK-3β in the liver tissue was also decreased (P<0.05) in the model mice, consistent with the results from the primary rat hepatocytes. Our results suggests that resistin reduces the levels of GSK-3β phosphorylated at Ser 9 leading to impaired hepatic insulin action in primary rat hepatocytes and in a mouse model with high liver-specific expression of resistin.
PMCID: PMC3897445  PMID: 23860320 CAMSID: cams3833
Resistin; Insulin resistance; Glycogen synthase kinase-3β (GSK-3β); Liver
19.  N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification 
Gene  2013;529(1):27-36.
Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification.
PMCID: PMC3805111  PMID: 23941992
apigenin; apoptosis; differentiation; neuroblastoma; N-Myc shRNA
20.  Characterization of the Human Mitochondrial Thiamine Pyrophosphate Transporter SLC25A19 Minimal Promoter: a Role for NF-Y in Regulating Basal Transcription 
Gene  2013;528(2):248-255.
Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5′-regulatory region of the SLC25A19 gene (1,080 bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5′- and 3′-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between −131 and + 20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.
PMCID: PMC3764993  PMID: 23872534
SLC25A19; promoter; transcription regulation; thiamine pyrophosphate; mitochondria
21.  Detecting novel genes with sparse arrays 
Gene  2010;467(0):41-51.
Species-specific genes play an important role in defining the phenotype of an organism. However, current gene prediction methods can only efficiently find genes that share features such as sequence similarity or general sequence characteristics with previously known genes. Novel sequencing methods and tiling arrays can be used to find genes without prior information and they have demonstrated that novel genes can still be found from extensively studied model organisms. Unfortunately, these methods are expensive and thus are not easily applicable, e.g., to finding genes that are expressed only in very specific conditions.
We demonstrate a method for finding novel genes with sparse arrays, applying it on the 33.9 Mb genome of the filamentous fungus Trichoderma reesei. Our computational method does not require normalisations between arrays and it takes into account the multiple-testing problem typical for analysis of microarray data. In contrast to tiling arrays, that use overlapping probes, only one 25mer microarray oligonucleotide probe was used for every 100 b. Thus, only relatively little space on a microarray slide was required to cover the intergenic regions of a genome. The analysis was done as a by-product of a conventional microarray experiment with no additional costs. We found at least 23 good candidates for novel transcripts that could code for proteins and all of which were expressed at high levels. Candidate genes were found to neighbour ire1 and cre1 and many other regulatory genes. Our simple, low-cost method can easily be applied to finding novel species-specific genes without prior knowledge of their sequence properties.
PMCID: PMC4175568  PMID: 20691772
Microarray; Gene prediction; Moulds; Transcript
22.  A Current Review of Molecular Mechanisms Regarding Osteoarthritis and Pain 
Gene  2013;527(2):440-447.
Osteoarthritis afflicts millions of individuals across the world resulting in impaired quality of life and increased health costs. To understand this disease, physicians have been studying risk factors, such as genetic predisposition, aging, obesity, and joint malalignment; however have been unable to conclusively determine the direct etiology. Current treatment options are short-term or ineffective and fail to address pathophysiological and biochemical mechanisms involved with cartilage degeneration and the induction of pain in arthritic joints. OA pain involves a complex integration of sensory, affective, and cognitive processes that integrate a variety of abnormal cellular mechanisms at both peripheral and central (spinal and supraspinal) levels of the nervous system Through studies examined by investigators, the role of growth factors and cytokines has increasingly become more relevant in examining their effects on articular cartilage homeostasis and the development of osteoarthritis and osteoarthritis-associated pain. Catabolic factors involved in both cartilage degradation in vitro and nociceptive stimulation include IL-1, IL-6, TNF-α, PGE2, FGF-2 and PKCδ, and pharmacologic inhibitors to these mediators, as well as compounds such as RSV and LfcinB, may potentially be used as biological treatments in the future. This review explores several biochemical mediators involved in OA and pain, and provides a framework for the understanding of potential biologic therapies in the treatment of degenerative joint disease in the future.
PMCID: PMC3745800  PMID: 23830938
Osteoarthritis; cartilage; biochemical mediators; osteoarthritic pain
23.  A novel deletion mutation in the proton-coupled folate transporter (PCFT; SLC46A1) in a Nicaraguan child with hereditary folate malabsorption 
Gene  2013;527(2):10.1016/j.gene.2013.06.039.
Hereditary Folate Malabsorption (OMIM 229050) is a rare autosomal recessive disorder caused by loss-of-function mutations in the proton-coupled folate transporter gene (PCFT/SLC46A1) resulting in impaired folate transport across the intestine and into the central nervous system. We report a novel, homozygous, deletion mutation in a child of Nicaraguan descent in exon 2 (c.558-588del, ss778190447) at amino acid position I188S resulting in a frame shift with a premature stop.
PMCID: PMC3836604  PMID: 23816405
PCFT, proton-coupled folate transporter; HCP1, heme carrier protein; HFM, hereditary folate malabsorption; Folates; Intestinal folate transport; Folate deficiency
24.  Dynamic expression of 3′ UTRs revealed by Poisson hidden Markov modeling of RNA-Seq: Implications in gene expression profiling 
Gene  2013;527(2):616-623.
RNA sequencing (RNA-Seq) allows for the identification of novel exon-exon junctions and quantification of gene expression levels. We show that from RNA-Seq data one may also detect utilization of alternative polyadenylation (APA) in 3′ untranslated regions (3′ UTRs) known to play a critical role in the regulation of mRNA stability, cellular localization and translation efficiency. Given the dynamic nature of APA, it is desirable to examine the APA on a sample by sample basis. We used a Poisson hidden Markov model (PHMM) of RNA-Seq data to identify potential APA in human liver and brain cortex tissues leading to shortened 3′ UTRs. Over three hundred transcripts with shortened 3′ UTRs were detected with sensitivity >75% and specificity >60%. tissue-specific 3′ UTR shortening was observed for 32 genes with a q-value ≤ 0.1. When compared to alternative isoforms detected by Cufflinks or MISO, our PHMM method agreed on over 100 transcripts with shortened 3′ UTRs. Given the increasing usage of RNA-Seq for gene expression profiling, using PHMM to investigate sample-specific 3′ UTR shortening could be an added benefit from this emerging technology.
PMCID: PMC3902974  PMID: 23845781
Untranslated region; Alternative polyadenylation; Microarray
25.  Prolonged fasting activates hypoxia inducible factor -1α, -2α and -3α in a tissue-specific manner in northern elephant seal pups 
Gene  2013;526(2):155-163.
Hypoxia inducible factors (HIFs) are important regulators of energy homeostasis and cellular adaptation to low oxygen conditions. Northern elephant seals are naturally adapted to prolonged periods (1–2 months) of food deprivation (fasting) that result in metabolic changes that may activate HIF-1. However, the effects of prolonged fasting on HIFs are not well defined. We obtained the full-length cDNAs of HIF-1α and HIF-2α, and partial cDNA of HIF-3α in northern elephant seal pups. We also measured mRNA and nuclear protein content of HIF-1α, -2α, -3α in muscle and adipose during prolonged fasting (1, 3, 5 & 7 wks), along with mRNA expression of HIF-mediated genes, LDH and VEGF. HIF-1α, -2α and -3α are 2595, 2852 and 1842 bp and encode proteins of 823, 864 and 586 amino acid residues with conserved domains needed for their function (bHLH and PAS) and regulation (ODD and TAD). HIF-1α and -2α mRNA expression increased 3- to 5-fold after 7 weeks of fasting in adipose and muscle, whereas HIF-3α increased 5-fold after 7 weeks of fasting in adipose. HIF-2α protein expression was detected in nuclear fractions from adipose and muscle, increasing approximately 2-fold, respectively with fasting. Expression of VEGF increased 3-fold after 7 weeks in adipose and muscle, whereas LDH mRNA expression increased 12-fold after 7 weeks in adipose. While the 3 HIFα genes are expressed in muscle and adipose, only HIF-2α protein was detectable in the nucleus suggesting that HIF-2α may contribute more significantly in the up-regulation of genes involved in the metabolic adaption during fasting in the elephant seal.
PMCID: PMC3729614  PMID: 23707926
hypoxia inducible factor; northern elephant seal; gene expression; prolonged fasting; nuclear protein

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