Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and over-expressed in many cancers. The cyclin D1b mRNA variant was established in human cells and recent functional analyses revealed that its protein product harbors unique activities in human cancer cells. By performing reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) experiments, we identified the cyclin D1b mRNA variant in mouse. Similar to its human counterpart, the mouse cyclin D1b transcript consists of exon 1, 2, 3, 4 and part of intron 4, and contains a long open reading frame (ORF). The predicted peptide from this ORF is 34-amino acid longer than the human cyclin D1b. The expression of this mouse mRNA variant was investigated. It appears to be expressed ubiquitously and differentially in various mouse cell lines and tissues and its level might be proportional to that of the canonical endogenous cyclin D1a mRNA.
RT-PCR; RACE; Exon; Intron; DNA sequencing; Cell cycle
The acetylcholinesterase of Lepidoptera insects is encoded by two genes, ace1 and ace2. The expression of the ace1 gene is significantly higher than that of the ace2 gene, and mutations in ace1 are one of the major reasons for pesticide resistance in insects. In order to investigate the effects of the mutations in ace1’s characteristic sites on pesticide resistance, we generated mutations for three amino acids using site-directed mutagenesis, which were Ala(GCG)303Ser(TCG), Gly(GGA)329Ala(GCA) and Leu (TCT)554Ser(TTC). The Baculovirus expression system was used for the eukaryotic expression of the wild type ace1 (wace1) and the mutant ace1 (mace1). SDS-PAGE and Western blotting were used to detect the targeting proteins with expected sizeof about 76 kDa. The expression products were purified for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. We observed no significant differences in the overall activity of the wild type and mutant AChEs. However, with 10 min of physostigmine (10 μM) inhibition, the remaining activity of the wild type AChE was significantly lower than that of the mutant AChE. Ten min inhibition with 33.4 μM phoxim also resulted in significantly lower remaining activity of the wild type AChE than that of the mutant AChE. These results indicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine and phoxim, which laid the foundation for future in vivo studies on AChE’s roles in pesticide resistance.
Bombyx mori; Acetylcholinesterase; Gene mutation
Actin is one of the most abundant proteins in eukaryotic cells, where it plays key roles in cell shape, motility, and regulation. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerization of monomeric G-actin molecules through sequential rowing, is called F-actin. Recently, the crystal structure of an actin dimer has been reported, which details molecular interface in F-actin. In this study, the computational prediction model of actin and actin complex has been constructed base on the atomic model structure of G-actin. To this end, a docking simulation was carried out using predictive docking tools to obtain modeled structures of the actin–actin complex. Following molecular dynamics refinement, hot spots interactions at the protein interface were identified, that were predicted to contribute substantially to the free energy of binding. These provided a detailed prediction of key amino acid interactions at the protein–protein interface. The obtained model can be used for future experimental and computational studies to draw biological and functional conclusions. Also, the identified interactions will be used for designing next studies to understand the occurrence of F-actin structure.
F-actin; G-actin; Protein–protein interaction; Docking; Hot spots
One-third of all individuals with epilepsy are resistant to antiepileptic drug (AED) treatment. Antiepileptic treatment response has been suggested to be modulated by genetic polymorphisms of drug efflux transporters. Several polymorphic variants within the multidrug resistance 1 (MDR1) gene, which encodes the major transmembrane efflux transporter P-glycoprotein, have been proposed to be associated with AED resistance in epilepsy patients. The aim of this study was to evaluate the effect of C3435T and G2677T/A polymorphisms of MDR1 on AED resistance in Turkish children with epilepsy. MDR1 C3435T and G2677T/A were genotyped in 152 patients with epilepsy, classified as drug-resistant in 69 and drug-responsive in 83. Genotypes of the C3435T and G2677T/A polymorphisms were determined by polymerase chain reaction followed by restriction fragment length polymorphism. Genotype and allele frequencies of C3435T and G2677T/A polymorphisms of the MDR1 gene did not differ between drug-resistant and drug-responsive epilepsy patients. Our results suggest that MDR1 C3435T and G2677T/A polymorphisms are not associated with AED resistance in Turkish epileptic patients. To clarify the exact clinical implication of the MDR1 polymorphisms on the multidrug resistance in epilepsy, further investigations in various ethnic populations would be necessary.
Drug resistance; Epilepsy; MDR1; P-gp; Polymorphism
Signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) are transcription factors involved in cell survival, inflammation and metastasis. Constitutively activated STAT3 is found in many cancers, including melanoma. To study the crosstalk between STAT3 and NFκB signaling and its role in regulation of cancer cell survival, we used RNA interference (RNAi) to down-regulate STAT3 expression in human melanoma cells. RNAi strategies including double-stranded RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA are widely used to knock down disease-causing genes in a targeted fashion. We found that shRNAs up-regulate non-specific NFκB activity, while siRNA directed against STAT3 specifically increase NFκB activity. The basal survival of melanoma cells is unaffected by STAT3 knockdown—likely due to activation of pro-survival NFκB signaling. Whereas, owing to off-target effects, plasmid-transcribed shRNA affects melanoma survival. Our data show that shRNA-mediated gene silencing induces non-specific or off-target effects that may influence cell functions.
Electronic supplementary material
The online version of this article (doi:10.1007/s11033-013-2817-7) contains supplementary material, which is available to authorized users.
Transcription factor STAT3; STAT3-NFκB crosstalk; Cancer cell survival; RNAi
Cytokines, involved in the T-helper 1 system, play a role in the regulation of hepatitis B virus (HBV) clearance and the immune response to HBV antigens during natural infection or planned vaccination. Our aim was to examine whether the polymorphic variants of IL-12 are equally associated with development of antibodies to HBV surface antigen (anti-HBs) in hemodialysis (HD) patients in the case of HBV vaccination or HBV infection. The IL-12A rs568408 and IL-12B rs3212227 polymorphisms were analyzed in relation to anti-HBs development in 602 HD patients with negative antibodies to HBV core antigen (anti-HBc) who were hepatitis B vaccinated (group I) as well as in 237 anti-HBc positive HD patients who were infected with HBV in the past (group II). In group I, 199 patients did not develop an anti-HBs titre >10 IU/L (subgroup Ia), whereas in group II, 55 patients did not develop an anti-HBs titre >10 IU/L (subgroup IIa). Patients of groups I and II that developed an anti-HBs >10 IU/L were included into subgroups Ib and IIb, respectively. In hepatitis B vaccinated HD patients, development of a protective anti-HBs titre was positively associated with vintage of renal replacement therapy (RRT), chronic glomerulonephritis as a cause of RRT, and GA rs 568408 IL-12A (OR 1.6, 95 % CI 1.0–2.5, P = 0.035), but a frequency distribution of this genotype between responders and non-responders was not significant when the Bonferroni correction was applied. In HBV infected HD patients, anti-HBs development was positively associated with AC rs3212227 IL-12B (OR 8.0, 95 % CI 2.6–24.9, P < 0.001), whereas HBsAg positivity, AA rs3212227 IL-12B (OR 0.3, 95 % CI 0.1–0.7, P = 0.007), and CC rs3212227 IL-12B (OR 0.1, 95 % CI 0.03–0.6, P = 0.011) were negative predictors of positive anti-HBs phenotype. When the Bonferroni correction was applied, if appropriate, these associations remained significant. In HD patients, the studied IL-12 polymorphic variants seem to be associated with the anti-HBs phenotype (a) with borderline significance for IL-12A in hepatitis B vaccinated patients, and (b) significantly for IL-12B in patients who underwent natural HBV infection.
Anti-HBs; Gene polymorphism; Hemodialysis; Infection; Interleukin-12; Vaccination
Polymorphic variants at the interleukin-2 (IL2) locus affect the risk of several autoimmune disorders. Our aim was to evaluate the association of the four IL2 polymorphisms (rs6822844, rs6534349, rs2069762 and rs3136534) with type 1 diabetes (T1D) in the Polish population, and to correlate them with the serum interleukin-2 levels. 543 unrelated T1D patients and 706 healthy control subjects were enrolled. The minor T allele at rs6822844 was significantly less frequent in T1D compared to controls (p = 0.002; OR 0.71; 95 % CI 0.571–0.880). Likewise, the frequency of the TT genotype was decreased among the affected individuals (p = 0.007). In healthy subjects, stratification according to the rs6822844 genotype revealed significant differences in circulating interleukin-2 (p = 0.037) with the highest levels in TT protective genotypes. Three other IL2 polymorphisms did not display significant differences in allele and genotype distribution. In conclusion, the rs6822844 variant is associated with T1D and may play a functional role, or reflect the influence of another causative genetic variant in linkage disequilibrium.
IL2 gene; Polymorphism; Interleukin-2; Type 1 diabetes
The predisposing role to human obesity of the MC3R gene polymorphism is controversial. In this report we present the first study focused on the search for the MC3R polymorphism in the Polish population. Altogether 257 obese children and adolescents (RBMI>120) and 94 adults, who were never obese or overweight (BMI<25), were studied. For all subjects the entire coding sequence was analyzed by direct DNA sequencing. One common polymorphism (81Val>Ile) and two rare mutations (257Arg>Ser and 335Ile>Ser) were identified. The common polymorphism was widely distributed in the obese and control cohorts, while the mutations were identified in four obese subjects only. In case of the 335Ile>Ser substitution a three-generation family, consisting of 20 members, was also analyzed. It was found that all carriers of the 335Ser mutation were obese, but among non-carriers obese subjects also were found. Our study suggests that the predisposing effect to obesity of the 81Ile polymorphic variant is rather unlikely. With regard to the studied rare mutations we suggest that the 335Ser allele may have a small predisposing effect.
Electronic supplementary material
The online version of this article (doi:10.1007/s11033-013-2808-8) contains supplementary material, which is available to authorized users.
Human; Obesity; MC3R gene; Polymorphism; Mutation
Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.
Exopalaemon carinicauda; Peroxiredoxin 5 (Prx5); Gene cloning; Expression
In lung cancer pathogenesis, genetic instability, i.e., loss of heterozygosity (LOH) and microsatellite instability (MSI) is a frequent molecular event, occurring at an early stage of cancerogenesis. The presence of LOH/MSI in non-small cell lung carcinoma (NSCLC) was found in many chromosomal regions, but exclusive of 3p their diagnostic value remains controversial. In this study we focused on other than 3p regions—1p31.2, 7q32.2, 9p21.3, 11p15.5, 12q23.2 and 16q22—the loci of many oncogenes and tumour suppressor genes. To analyze the potential role of LOH/MSI involved in NSCLC pathogenesis we allelotyped a panel of 13 microsatellite markers in a group of 56 cancer specimens. Our data demonstrate the presence of allelic loss for all (13) analyzed markers. Total LOH/MSI frequency in NSCLC was the highest for chromosomal region 11p15.5 (25.84 %), followed by 9p21.3 and 1p31.2 (19.87 and 16.67 % respectively). A statistically significant increase of total LOH/MSI frequency was detected for the 11p15.5 region (p = 0.0301; χ2 test). The associations of total LOH/MSI frequency: 1) increase in 11p15.5 region (p = 0.047; χ2 test) and 2) decrease in 7q32.2 region (p = 0.037; χ2 test) have been statistically significant in AJCC III (American Joint Committee on Cancer Staging). In Fractional Allele Loss (FAL) index analysis, the correlation with cigarette addiction has been statistically significant. The increased amount of cigarettes smoked (pack years) in a lifetime correlates with increasing FAL (p = 0.024; Kruskal–Wallis test). These results demonstrate that LOH/MSI alternation in studied chromosomal regions is strongly influenced by tobacco smoking but do not seem to be pivotal NSCLC diagnostic marker with prognostic impact.
Non-small cell lung carcinoma (NSCLC); Loss of heterozygosity (LOH); Microsatellite instability (MSI); Microsatellite markers; Genetic instability
The physiological, biochemical and molecular mechanisms regulating the initiation of a regenerative pathway remain partially unknown. Efforts to identify the biological features that confer transformation ability, or the tendency of some cells to induce transgene silencing, would help to improve plant genetic engineering. The objective of our study was to monitor the evolution of plant cell competencies in relation to both in vitro tissue culture regeneration and the genetic transformation properties. We used a simple wheat regeneration procedure as an experimental model for studying the regenerative capacity of plant cells and their receptivity to direct gene transfer over the successive steps of the regenerative pathway. Target gene profiling studies and biochemical assays were conducted to follow some of the mechanisms triggered during the somatic-to-embryogenic transition (i.e. dedifferentiation, cell division activation, redifferentiation) and affecting the accessibility of plant cells to receive and stably express the exogenous DNA introduced by bombardment. Our results seem to indicate that the control of cell-cycle (S-phase) and host defense strategies can be crucial determinants of genetic transformation efficiency. The results from studies conducted at macro-, micro- and molecular scales are then integrated into a holistic approach that addresses the question of tissue culture and transgenesis competencies more broadly. Through this multilevel analysis we try to establish functional links between both regenerative capacity and transformation receptiveness, and thereby to provide a more global and integrated vision of both processes, at the core of defense/adaptive mechanisms and survival, between undifferentiated cell proliferation and organization.
Electronic supplementary material
The online version of this article (doi:10.1007/s11033-013-2696-y) contains supplementary material, which is available to authorized users.
Developmental window; In vitro tissue culture; Genetic transformation; Mature embryo; Regeneration; Somatic-to-embryogenic transition
To explore the anti-tumor effect and immune mechanism mediated by a new recombinant adeno-associated virus (rAAV) encoding secondary lymphoid tissue chemokine (SLC) mature peptide gene. AAV Helper-Free system was used for rAAV-SLC package. The anti-tumor effect of SLC was detected by bearing tumor established from Hepal-6 cells both in C57BL/6J and nude mice. Flow cytometry analysis and IHC for Tumor-infiltrating T cells and CD11c+DCs were also investigated to explore the immunological mechanism. rAAV-SLC was successfully packaged in AAV293 cells and transfected Hepal-6 tumor cells at high efficiency. The anti-tumor effect was demonstrated by less tumor weight and longer survival outcome. Coincident with the anti-tumor response, local elaboration of SLC within the tumor bed elicited a heavy infiltration of CD4+, CD8+T cells and CD11c+ dendritic cells into the tumor sites. More importantly, there was higher infiltration of Foxp3+ regulatory T cells (Tregs). Local elaboration of SLC mediated by rAAV-SLC has strong T cell mediated anti-tumor effect. The study also suggested that Tregs in the tumor microenvironment tampered the anti-tumor effect.
Recombinant adeno-associated virus; Secondary lymphoid tissue chemokine; Anti-tumor effect; Tregs
Mitochondrial respiratory chain deficiencies are a group of more than 100 disorders of adults and children, with highly variable phenotypes. The high prevalence of mitochondrial disorders (MIDs) urges the clinician to diagnose these disorders accurately, which is difficult in the light of highly variable and overlapping phenotypes, transmission patterns and molecular backgrounds. Fibroblast growth factor 21 (FGF-21) is an important endocrine and paracrine regulator of metabolic homeostasis. The FGF-21 transcript is reported to be abundantly expressed in liver, but little is known about the regulation of FGF-21 expression in other tissues. FGF-21 could play a role in the metabolic alterations that are often associated with mitochondrial diseases. The aim of this study was to show the association of the FGF-21 biomarker with human primary MIDs and secondary MIDs in suspected patients in Iran. Serum FGF-21 levels were determined using ELISA in 47 mitochondrial patients, including 32 with primary MIDs, 15 patients with Friedreich ataxia as a secondary MID and 30 control subjects. Serum FGF-21 levels were significantly higher in subjects with the primary MIDs (p < 0.05), compared to subjects without MIDs. However, serum FGF-21 levels did not show significant increase in subjects with FA as a secondary MID. There is an association between increasing concentrations of FGF-21 with mitochondrial diseases, suggesting FGF-21 as a biomarker for diagnosis of primary MIDs in humans. However, this biomarker is not appropriate for the diagnosis of FA.
Fibroblast growth factor 21 (FGF-21); Respiratory chain deficiency (RCD); Mitochondrial disorders (MIDs); Friedreich ataxia (FA); OXPHOS
Iron can play a role in colorectal cancer (CRC) development. The expression of genes involved in iron metabolism and its regulation in CRC has not been investigated well. Also the correlation between the level of iron-related genes expression and cancer progression is not known. In this study we collected paired samples of primary adenocarcinoma and adjacent normal mucosa from 73 patients. We assessed the mRNA or miRNA levels of 21 genes and verify their association with clinicopathological characteristics of CRC patients. Our experiments revealed, that the level of divalent metal transporter 1 transcript is well correlated with mRNA levels of iron regulatory proteins (IRPs) in tumor specimens. We have shown, that IRP2 can also be engaged in the mRNA stabilization of other iron transporter–transferrin receptor 1 (TfR1) in early stage of disease, however, in more advanced stages of CRC, mRNA level of TfR1 is related to miR-31 level. For the first time we have shown, that ferroportin concentration is significantly associated with miR-194 level, causing the reduction of this transporter amount in tumor tissues of patients with more advanced stages of CRC. We have also shown the alterations in expressing profile of miR-31, miR-133a, miR-141, miR-145, miR-149, miR-182 and miR-194, which were observed even in the early stage of disease, and identified a set of genes, which take place in correct assigning of patients in dependence of CRC stage. These iron-related genes could become potential diagnostic or prognostic indicators for patients with CRC.
Colorectal adenocarcinoma; Iron metabolism; MiRNA expressing profile; Diagnosis of cancer
Two-dimensional electrophoresis, coupled with MALDI-TOF-MS, was used to identify differentially expressed proteins between young and mature leaves of sweet potato [Ipomoea batatas (L.) Lam]. The results showed that there were 25 differential proteins between young and mature leaves. The Rubisco activase (RCA) that catalyzes the activation of Rubisco in vivo and plays a crucial role in photosynthesis was among these 25 proteins. So far, little was known about the molecular biology of RCA in sweet potato. Here, this research reports the cloning and characterization of two genes encoding the short isoform and the long isoform of sweet potato RCAs. Analysis of DNA sequences of RCA suggested that the corresponding mRNAs were transcribed from two different genes. To study the roles of these two RCA isoforms in photosynthesis, we investigated the expression patterns of these RCA genes at the mRNA and protein levels every 2 h in a photoperiod and under different temperatures conditions. The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both. In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl–RCAs–RCAl), at least in yeast cells. These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.
Electronic supplementary material
The online version of this article (doi:10.1007/s11033-013-2744-7) contains supplementary material, which is available to authorized users.
Gene expression; Protein interaction; Rubisco activase; Sweet potato; Two-dimensional electrophoresis
Immense body of evidence indicates that dysfunction of immune system is implicated in the etiology of schizophrenia. The immune theory of schizophrenia is supported by alterations in cytokine profile in the brain and peripheral blood. Given the strong genetic background of schizophrenia, it might be assumed that aberrant production of cytokines might be the consequence of genetic factors. This study aimed at investigating the association between schizophrenia susceptibility and selected functional polymorphisms in genes encoding cytokines including: interleukin-2 (IL2 −330T>G, rs2069756), interleukin-6 (IL-6 −174G>C, rs1800795), interferon-γ (IFNG +874T>A, rs2430561) as well as for the first time transforming growth factor-β1 (TGFB1 +869T>C, rs1800470 and +916G>C, rs1800471). We recruited 151 subjects with schizophrenia and 279 controls. There was a significant difference in the genotype distribution and allelic frequency of the TGFB1 +869T>C between patients with schizophrenia and healthy controls (p < 0.05). The risk of schizophrenia was more than two-fold higher in carriers of T allele (CT+TT genotypes) than individuals with CC genotype. Given documented gender differences in incidence of schizophrenia, we conducted separate analyses of male and female participants. We have shown that the association was significant in females, while in males it reached a trend toward statistical significance. To the best of our knowledge, it is the first report showing the association between TGFB1 +869T>C polymorphism and schizophrenia.
Schizophrenia; Interleukin; Transforming growth factor; Genetic polymorphism; Cytokine; Interferon
The importance of matrix metalloproteinase 8 (MMP8) expression during the progression of thoracic aortic dissection (TAD) has been recently emphasized. Genetic variations that affect proteinase expression or activity might contribute to the pathogenesis of TAD. In this study, we investigated whether the MMP8 C-799T genotype is associated with TAD. The frequency distributions of the MMP8 C-799T polymorphism were determined by direct sequencing. Associations between the polymorphism and disease progression in TAD were investigated. The level of plasma and tissue MMP8 was measured by enzyme-linked immunosorbent assay and western blotting. The MMP8 C-799T polymorphism was significantly associated with susceptibility to disease progression in TAD patients (n = 152) than in controls (n = 147) (P = 0.004, OR = 0.62, 95 % CI 0.45–0.86). The TT homozygotes had a significantly higher risk of TAD compared to C allele carriers in a logistic regression model, after adjustment for the conventional risk factors for TAD. The plasma MMP8 concentration was significantly higher in TAD patients compared to control patients (P < 0.05). TT genotypes had increased MMP8 levels compared to CC and CT genotype carriers in both TAD and control subjects (P < 0.05). The C-799T polymorphism in the MMP8 promoter is part of the genetic variation underlying the susceptibility of individuals to the progression of TAD.
Thoracic aortic dissection; MMP8; Polymorphism
A recent genome-wide association study elucidated that 4q25 was implicated in ischemic stroke, but subsequent studies showed inconsistent results. In order to get coincident conclusion, we investigated two SNPs (rs2200733, rs10033464) on chromosome 4q25 in 1,388 stroke patients and 1,629 controls from Chinese Han population and then performed a meta-analysis. Although we failed to detect any association between 4q25 and stroke in our case–control study, meta-analysis revealed that rs2200733 showed association with overall stroke (OR 1.18, 95 % CI 1.08–1.27), but not for rs10033464. Subsequently subgroup analysis indicated that both rs2200733 and rs10033464 conferred increased risk for cardioembolic stroke (CE stroke) (for rs2200733, OR 1.38, 95 % CI 1.26–1.51; for rs10033464, OR 1.14, 95 % CI 1.02–1.26), while rs2200733 was marginal associated with non-CE stroke (OR 1.09, 95 % CI 1.02–1.16). our results demonstrated that two SNPs (rs2200733 and rs1003346) on chromosome 4q25 were limited to the stroke of cardioembolic etiology. To confirm this conclusion, well-designed studies with larger sample size involving case–control populations with homogeneous ancestry warrant to be conducted in the future.
Electronic supplementary material
The online version of this article (doi:10.1007/s11033-013-2707-z) contains supplementary material, which is available to authorized users.
Stroke; 4q25; Meta-analysis
While most Japanese apricot (Prunus mume Sieb. et Zucc.) cultivars display typical S-RNase-based gametophytic self-incompatibility, some self-compatible (SC) cultivars have also been identified. In this study, we confirmed SC of ‘Zaohong’ through replicated self-pollination tests. Cross-pollination tests showed that SC of ‘Zaohong’ was caused by a loss of pollen function, so we determined that the S-genotype of ‘Zaohong’ was S2S15. Sequence analysis of the S-haplotypes of ‘Zaohong’ showed no mutations which were likely to alter gene function. Furthermore, expression analysis based on RT-PCR of S-locus genes revealed no differences at the transcript level when compared with ‘Xiyeqing’, a self-incompatible cultivar with the same S haplotypes. In addition, except for S-locus genes, a new type of F-box gene encoding a previously uncharacterised protein with high sequence similarity (61.03–64.65 %) to Prunus SFB genes was identified. Putative structural regions of PmF-box genes have been described, corresponding to regions in PmSFB alleles, but with some sequence variations. These results suggest that SC in ‘Zaohong’ occurs in pollen, and that other factors outside the S-locus, including PmF-box genes, might be associated with the loss of function of pollen S genes.
Japanese apricot; Self-compatibility (compatible) (SC); Self-incompatibility (incompatible) (SI); S-RNase; SFB; PmF-box
It has been shown that human and murine fibroblasts can be reprogrammed by ectopic expression of transcription factors using viral vectors. For the purpose of human therapeutic applications, the integration of viral transgenes into the genome is unlikely to be accepted. We therefore produced recombinant transcription factor proteins in E. coli (OCT4, SOX2, c-MYC and KLF4) carrying the cell penetrating TAT domain from HIV1. The purified proteins were able to enter into mammalian cells when added to tissue culture medium but appeared not to translocate to the nucleus. Further investigation indicated that most of the protein was tied up in the endosomes and was unavailable for reprogramming. Once this problem has been solved it seems likely that protein reprogramming will be the method of choice for clinical applications.
Induced pluripotent stem cells (iPSCs); TAT peptide; Reprogramming; Transcription factors; Gene expression
This study aimed to explore the protective effect of hydrogen as an antioxidant on monocrotaline (MCT)-induced pulmonary hypertension (PH). Forty-eight SD rats were equally randomized into four groups: SHAM group, MCT group, MCT+Oral-H2 group and MCT+Inj-H2 group. The results showed that the mean pulmonary arterial pressure, right ventricle weight and right ventricular hypertrophy index in MCT group were significant higher than those in SHAM group; pulmonary inflammatory response, atrial natriuretic factor, 3-nitrityrosine and intercellular adhesion molecule-1 were also increased significantly in MCT group. These indexes were decreased significantly in both MCT+Oral-H2 group and MCT+Inj-H2 group, which indicate Oral-H2 and Inj-H2 have similar effects of preventing the development of PH and mitigating RV hypertrophy. The protective effect of hydrogen is associated with its antioxidative ability and action of reducing pulmonary inflammatory response. While Oral-H2 is more convenient than Inj-H2, Oral-H2 may be ideal for clinical use in future.
Antioxidant; Hydrogen water; Pulmonary hypertension
The Zinc Finger Homeodomain Enhancer-binding Protein (Zfhep) is involved in skeletal patterning, immune cell, muscle, and brain development, and is necessary for life. Zfhep contains a single central homeodomain (HD) adjacent to an isolated zinc finger, the function of which is unknown. The placement of a zinc finger so close to a homeodomain is novel in nature. The aim of this work was to characterize the Zfhep homeodomain (HD) or the zinc finger homeodomain (ZHD), with respect to DNA-binding and protein-protein interactions. Glutathione-S-transferase (GST) fusion proteins containing either just the HD or both the zinc finger and HD (ZHD) were expressed in E. coli. The GST fusion protein affinity-binding assay demonstrated that Zfhep ZHD interacts specifically with the POU domain of the Oct-1 transcription factor. The adjacent zinc finger is required since Zfhep HD alone does not interact with Oct-1 POU domain. Furthermore, ZHD does not bind to the POU homeodomain lacking the POU specific region. These results demonstrate that the Zfhep zinc finger homeodomain motif functions as a protein-binding domain in vitro, and suggests that Zfhep may modulate the activity of POU domain transcription factors. However, neither the Zfhep ZHD nor the HD bound DNA in EMSA or selected a DNA-binding site from a pool of random oligonucleotides. This is the first demonstration of a function for the HD region of Zfhep, which is the first case of a bi-partite domain requiring both a zinc finger and a HD for binding to protein.
AREB6; Oct-1; POU domain; Zfhep; ZEB; deltaEF1
The Investigator DIPplex® kit (Qiagen) contain components for the simultaneous amplification and analysis of 30 biallelic autosomal INDELs and amelogenin. The objective of this study was to estimate the diversity of the 30 markers in Polish (NP = 122) and Taiwanese (NT = 126) population samples and to evaluate their usefulness in forensic genetics. All amplicon lengths were shorter than 160 base pairs. The DIPplex genotype distributions showed no significant deviation from Hardy–Weinberg rule expectations (Bonferroni corrected) except for DLH39 in the Taiwanese population. Among the Poles and the Taiwanese the mean observed heterozygosity values are 0.4385 and 0.4079, and the combined matching probability values are 7.98 × 10−14 and 1.22 × 10−11, respectively. The investigated marker set has been confirmed as a potential extension to standard short tandem repeat-based kits or a separate informative system for individual identification and kinship analysis. Eight INDELs have been selected as possible ancestry informative single-nucleotide polymorphisms for further analyses.
INDEL polymorphism; Investigator DIPplex; Poland; Taiwan; Population genetics; Forensic efficiency
The aim of the study was to analyze the consequence of silencing genes coding for the key subunits of the telomerase complex, i.e. TERT, TERC and TP1 in human breast cancer MCF7 and MDA-MB-231cells. The transfection was performed using Lipofectamine2000 and pooled siRNAs. The cytotoxic and/or antiproliferative effect of siRNA was measured by the SRB assay, the cell cycle was analysed by flow cytometry and DNA fragmentation by TUNEL analysis. Telomerase activity was assessed by TRAP, followed by PAGE and ELISA assays. Telomerase downregulation was also assessed using qPCR in order to estimate the changes in the expression profile of genes engaged in apoptosis. It was revealed that treatment of breast cancer cells with different siRNAs (100 nM) resulted in a cell type and time-dependent effects. The downregulation of telomerase subunits was followed by reduction of telomerase activity down to almost 60 % compared to control cells. However, a significant effect was only observed when the TERT subunit was downregulated. Its silencing resulted in a significant (p < 0.05) increase of apoptosis (over 10 % in MCF7 and about 5 % in MDA-MB-231 cells, corresponding to the Annexin V assay) and DNA fragmentation (almost 30 % in MCF7 and over 25 % in MDA-MB-231 cells). Interestingly, also several proapoptotic genes were induced after the downregulation of the key telomerase subunit, including Bax, Bik or caspase-1 and caspase-14, as well as NGFR and TNFSF10 which were upregulated twice and more.
Adenocarcinoma therapy; Breast cancer; Telomerase; Telomere
Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a common flavonoid in many types of plants and has several beneficial biological effects, including anti-inflammation, anti-oxidant, and anti-cancer properties. However, the detail mechanisms of luteolin in suppressing tumor invasion and metastasis are poorly understood. Here, we investigated the effects of luteolin on suppressing glioblastoma tumor cell invasion and migration activity. Under the non-cytotoxic doses (15 and 30 μM), luteolin exhibited an inhibitory effect on migration and invasion in U-87 MG and T98G glioblastoma cells. Additionally, filopodia assembly in U-87 MG cells was markedly suppressed after luteolin treatment. The treatment of luteolin also showed a decrease of Cdc42 (cell division cycle 42) protein levels and reduced PI3K/AKT activation, whereas there was no association between this decrease and phosphorylated ERK or altered transcription levels of Cdc42. Over expression of constitutive Cdc42 (Q61L) using transient transfection in U-87 MG cells induced a partial cell migration, but did not affected the degradation of the protein levels of Cdc42 after luteolin treatment. Moreover, inhibition of the proteaosome pathway by MG132 caused a significant recovery in the migration ability of U-87 MG cells and augmented the Cdc42 protein levels after luteolin treatment, suggesting that pharmacological inhibition of migration via luteolin treatment is likely to preferentially facilitate the protein degradation of Cdc42. Taken together, the study demonstrated that flavonoids of luteolin prevent the migration of glioblastoma cells by affecting PI3K/AKT activation, modulating the protein expression of Cdc42 and facilitating their degradation via the proteaosome pathway.
Glioblastoma; Migration; Luteolin; PI3K/AKT; Cdc42; Proteasome degradation