BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a ¹⁶⁹QACRG¹⁷³ sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells.     

A duplex real-time PCR assay for detection of mutT4 and mutT2 mutations in Mycobacterium tuberculosis of Beijing genotype

To determine the polymorphism of mutT genes of Mycobacterium tuberculosis of Beijing genotype, we developed a duplex real-time PCR assay based on hybridization probes for the Roche LightCycler instrument. The assay rapidly detects mutations at codons 48 and 58 of genes mutT4 and at mutT2, respectively.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.     

Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea

Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

Tumour-suppressive microRNA-224 inhibits cancer cell migration and invasion via targeting oncogenic TPD52 in prostate cancer

Our recent study of the microRNA expression signature of prostate cancer (PCa) revealed that microRNA-224 (miR-224) is significantly downregulated in PCa tissues. Here, we found that restoration of miR-224 significantly inhibits PCa cell migration and invasion. Additionally, we found that oncogenic TPD52 is a direct target of miR-224 regulation. Silencing of the TPD52 gene significantly inhibits cancer cell migration and invasion. Moreover, TPD52 expression is upregulated in cancer tissues and negatively correlates with miR-224 expression. We conclude that loss of tumour-suppressive miR-224 enhances cancer cell migration and invasion in PCa through direct regulation of oncogenic TPD52.     

The epigenetic silencing of LIMS2 in gastric cancer and its inhibitory effect on cell migration

Recent finding has shown that LIMS2 (also known as PINCH2) functions as a natural regulator of the LIMS1-ILK-parvin complex formation and is associated with cell spreading and migration via integrins at focal adhesions. Here, we report for the first time the epigenetic silencing of LIMS2 in gastric tumors. Downregulation of LIMS2 was detected in 91% (10 of 11) of gastric cancer cell lines by real-time quantitative RT-PCR and 80% (8 of 10) of the LIMS2-downregulated cell lines were associated with CpG island hypermethylation at a 5'-upstream region of LIMS2. Furthermore, LIMS2 was restored in its non-expressing cell lines after treatment with 5-Aza-dC and/or trichostatin A. Loss of expression of LIMS2 was also detected in 53% (51 of 96) of primary gastric tumors. This decrease in expression level significantly correlated with an increase of the CpG island hypermethylation. In addition, the methylation status in any normal-appearing gastric tissues was gradually increased in an age-dependent manner, suggesting that the positive methylation in normal-appearing gastric mucosa can be due to 'field cancerization effect' as an early event in gastric carcinogenesis. Moreover, the transient transfection of LIMS2-siRNA significantly stimulated cell migration in gastric cancer cells but had no effects on cell growth. These results suggest that the frequent inactivation of LIMS2 by epigenetic alteration in gastric cancer may be important in tumor progression events, such as invasion and metastasis. Thus, LIMS2 may be useful as a molecular biomarker and a therapeutic target by increasing its expression and activity in gastric cancer.     

Identification of one novel mutation in the EVC2 gene in a Chinese family with Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is a rare autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. It is caused by biallelic mutations in the EVC or EVC2 gene. Here, we identified a novel nonsense mutation p.W828X (c.2484G>A) in exon 14 and a recurrent nonsense mutation p. R399X (c.1195C>T) in exon 10 of EVC2 gene in a Chinese boy with EvC. Identification of a novel genotype in EvC will provide clues to the phenotype–genotype relations and may assist not only in the clinical diagnosis of EvC but also in the interpretation of genetic information used for prenatal diagnosis and genetic counseling.     

Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee

The incidence of nosemosis has increased in recent years due to an emerging infestation of Nosema ceranae in managed honey bee populations in much of the world. A real-time PCR assay was developed to facilitate detection and quantification of both Nosema apis and N. ceranae in both single bee and pooled samples. The assay is a multiplexed reaction in which both species are detected and quantified in a single reaction. The assay is highly sensitive and can detect single copies of the target sequence. Real-time PCR results were calibrated to spore counts generated by standard microscopy procedures. The assay was used to assess bees from commercial apiaries sampled in November 2008 and March 2009. Bees from each colony were pooled. A large amount of variation among colonies was evident, signifying the need to examine large numbers of colonies. Due to sampling constraints, a subset of colonies (from five apiaries) was sampled in both seasons. In November, N. apis levels were 1212 ± 148 spores/bee and N. ceranae levels were 51,073 ± 31,155 spores/bee. In March, no N. apis was detected, N. ceranae levels were 11,824 ± 6304 spores/bee. Changes in N. ceranae levels were evident among apiaries, some increasing and other decreasing. This demonstrates the need for thorough sampling of apiaries and the need for a rapid test for both detection and quantification of both Nosema spp. This assay provides the opportunity for detailed study of disease resistance, infection kinetics, and improvement of disease management practices for honey bees.     

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link.We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Evaluation of real-time PCR targeting hbb gene for Borrelia species identification

Several molecular methods have been employed for Borrelia species identification. Newly developed technology, real-time polymerase chain reaction (RT-PCR), combines simultaneous amplification, detection and differentiation of strains in one PCR run. The aim of the study was to perform and evaluate RT-PCR for Borreliaburgdorferi sensu lato species identification. Borrelia species identification was accomplished on 374 Borrelia strains using two approaches: 1.) MluI restriction of entire borrelial chromosome (MluI-large restriction fragment patterns, LRFP), and 2.) RT-PCR targeting hbb gene and specific melting temperature (Tm) detection. The results of the two molecular methods were compared. With MluI-RFLP we were able to differentiate all Borrelia species and their subtypes within particular species. RT-PCR based on Tm determination identified unique strains within the species Borreliaafzelii (Tm 66.11 °C), B. burgdorferi sensu stricto (Tm 68.18 °C), Borreliaspielmanii (Tm 59.45 °C) and Borreliavalaisiana (Tm 59.62 °C). We were not able to distinguish the last two species that shared almost identical Tm. The large majority of Borreliagarinii strains shared Tm 51.42 °C, while subtype Mlg4 was characterized by Tm 56.87 °C. Strains of Borrelialusitaniae species also were heterogeneous; human isolate had Tm 63.47 °C while two tick isolates shared Tm 61.77 °C. Differences inside hbb gene enabled differentiation of the majority of Borrelia species, and revealed two clusters within B. garinii and B. lusitaniae species, respectively, but it was not possible to distinguish B. spielmanii form B. valaisiana. The major advantage of RT-PCR was that it was easy to perform and that the results were obtained within a few hours.     

High efficient gene targeting on the AGAMOUS gene in an ArabidopsisAtLIG4 mutant

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell.     

Helicobacter pylori causes runx3 gene methylation and its loss of expression in gastric epithelial cells, which is mediated by nitric oxide produced by macrophages

Previous reports have indicated that Helicobacter pylori (H. pylori) causes epigenetic changes of certain genes such as cancer suppression genes, which may be associated with carcinogenesis. However, the mechanism by which it causes epigenetic changes in certain genes and not in others is unclear. Presently, we focused on a cancer suppression gene, runx3, and demonstrated the following: (1) H. pylori induces nitric oxide (NO) production in macrophages. (2) NO causes methylation of runx3 in epithelial cells. (3) H. pylori induces the methylation of epithelial cells in the presence of macrophages, which is reversed by an NO-specific inhibitor. These results indicate that H. pylori-induced methylation is mediated by NO, and suggest that NO may be a key to the mechanism of how H. pylori causes epigenetic changes in certain genes. Additionally, we demonstrated that lipopolysaccharide, as well as H. pylori, induces NO-mediated methylation, indicating that other inflammation inducers beside H. pylori might induce aberrant methylation of runx3.     

br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development.