Characterization of Kudoa monodactyli n. sp (Myxosporea : Multivalvulida) from the muscle of Monodactylus argenteus (Teleostei : Monodactylidae) from Moreton Bay, Queensland, Australia

Kudoa monodactyli n. sp. is described from the somatic musculature of Monodactylus argenteus from several localities in southern Queensland, Australia. This is the first record of a myxozoan parasite from the family Monodactylidae. The spores typically have five polar capsules, making this species similar to the four other five-valved Kudoa species (K. neurophila, K. muscularis, K. shulmani, K. cutanea) that have been described to date. However, morphometric measurements particularly of spore length and width make the species from M. argenteus distinct from the other species. Comparison of the small subunit ribosomal DNA sequence of this species with its congeners for which sequence data are available, provides further evidence of novelty. Kudoa monodactyli n. sp. displays 38 (of 1,554) nucleotide differences compared with rDNA sequence of Kudoa neurophila, which on phylogenetic analysis places these species in clades exclusive of each other. Phylogenetic analyses also provide evidence that the number of valves per spore in this genus is an imperfect indicator of relatedness.

Genetic polymorphisms in post-mortem alcoholics

We investigated the hypothesis that alcoholism risk may be mediated by genes for neurotransmitters (dopamine, serotonin, opioid, GABAA and glutamate) associated with the dopamine reward system, and with genes involved in ethanol metabolism and fibrogenesis (ADH2, ADH3, ALDH2, CYP2E1, COL1A2, and ApoE). DNA was extracted from brain tissue collected at autopsy from pathologically characterised alcoholics and controls. PCR-based studies showed that alcoholism was associated with polymorphisms of the dopamine D2 receptor (DRD2) Taq1 B (p 0.005) and the GABAA 2 subunit C1412T (p 0.007) genes but not with the glutamate receptor subunit gene NR2B (366C/G), the serotonin transporter gene (5HTTL-PR), the dopamine transporter gene DAT1(SLC6A3), the Mu opioid receptor gene MOR1 (A118G and C1031G), the dopamine D2 receptor gene DRD2 Taq1 A or the GABAA 1(A15G), 6(T1519C) and 2(G3145A) subunit genes. The glial glutamate transporter gene EAAT2 polymorphism G603A was associated with alcoholic cirrhosis (p 0.024). The genotype for the most active alcohol dehydrogenase ADH3 was associated with a lower risk of alcoholism (p 0.027) and was less prevalent in alcoholics with DRD2 Taq1 A2/A2 (p 0.007), Taq1 B2/B2 (p 0.038) and GABAA-2 1412C/C (p 0.005) and EAAT2 603G/A (p 0.020) genotypes. Combined genotypes of DRD2 Taq1 A and B, GABAA-2, and EAAT2 G603A polymorphisms suggested a concerted influence of dopamine, GABAA and glutamatergic neurotransmitters in the predisposition to alcoholism.

Identification and localization of polyketide synthase genes from cultures of diarrheic shellfish poisoning toxin producing dinoflagellates of the genus Prorocentrum

Aquatic toxins are responsible for a number of acute and chronic diseases in humans. Okadaic acid (OA) and other dinoflagellate derived polyketide toxins pose serious health risks on a global scale. Ingestion of OA contaminated shellfish causes diarrheic shellfish poisoning (DSP). Some evidence also suggests tumor promotion in the liver by OA. Microcystin-LR (MC-LR) is produced by cyanobacteria and is believed to be the most common freshwater toxin in the US. Humans may be exposed to this acute hepatotoxin through drinking or recreational use of contaminated waters. ^ OA producing dinoflagellates have not been cultured axenically. The presence of associated bacteria raises questions about the ultimate source of OA. Identification of the toxin-producing organism(s) is the first step in identifying the biosynthetic pathways involved in toxin production. Polyketide synthase (PKS) genes of toxic and non-toxic species were surveyed by construction of clonal libraries from PCR amplicons of various toxic and non-toxic species of Prorocentrum in an effort to identify genes, which may be part of the biosynthetic pathway of OA. Analysis of the PKS sequences revealed that toxic species shared identical PKS genes not present in non-toxic species. Interestingly, the same PKS genes were identified in a library constructed from associated bacteria. ^ Subsequent bacterial small subunit RNA (16S) clonal libraries identified several common bacterial species. The most frequent 16S sequences found were identified as species of the genus Roseobacter which has previously been implicated in the production of OA. Attempts to culture commonly occurring bacteria resulted in the isolation of Oceanicaulis alexandrii , a novel marine bacterium previously isolated from the dinoflagellate Alexandrium tamarense, from both P. lima, and P. hoffmanianum. ^ Metabolic studies of microcystin-LR, were conducted to probe the activity of the major human liver cytochromes (CYP) towards the toxin. CYPs may provide alternate routes of detoxification of toxins when the usual routes have been inhibited. For example, some research indicates that cyanobacterial xenobiotics, in particular, lipopolysaccharides may inhibit glutathione S-transferases allowing the toxin to persist long enough to be acted upon by other enzymes. These studies found that at least one human liver CYP was capable of metabolizing the toxin. ^

Caracterização de um homólogo de HINT1 em cana-de-açúcar encontrado em bibliotecas subtrativas de CDNA para floração

The sugarcane is a monocot plant grown in tropical and subtropical regions, with Brazil being the largest producer. Despite its economic importance, little is known about the molecular flowering process in sugarcane. This physiological process can promote a loss up to 60% in sugar or bioethanol. Thus, this work had as objective characterize a HINT1 homologous gene previously identified in subtractive libraries of flowering. Genomic analysis of gene and promoter region structure allowed the observation that there are at least two distinct genes homologous to HINT on sugarcane. Bioinformatics analyses showed the conservation of the characteristic protein domain of HIT superfamily and indicate a phylogenetic relationship associated to cell location. Moreover, a possible relation with the SBTILISIN-like protein family through the information available in interatomas was observed. This suggests that the HINT gene of sugarcane can be related to plant development, there are several possibilities of interactions in the regulation of floral induction process, because the sequences present in regulatory regions indicate that differential expression of HINT was related to with climatic factors in the Northeast region of Brazil as well as to biotic stress and phytohormones. Furthermore, the sugarcane phenotypes indicate that the influence of HINT may happen due to product accumulation of its enzymatic activity. For these characteristics this gene can be used as a marker in the selection of new varieties.

New insights into Plagiogrammaceae (Bacillariophyta) based on multigene phylogenies and morphological characteristics with the description of a new genus and three new species.

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.

New insights into Plagiogrammaceae (Bacillariophyta) based on multigene phylogenies and morphological characteristics with the description of a new genus and three new species.

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

Background. One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1) and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1), an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results. The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap) alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions. We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used. © 2011 Tanzer et al; licensee BioMed Central Ltd.

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

Adenoviral gene therapy for non-small cell lung cancer

Adenoviral gene therapy is an experimental approach to cancer refractory to standard cancer therapies. Adenoviruses can be utilized as vectors to deliver therapeutic transgenes into cancer cells, while gene therapy with oncolytic adenoviruses exploits the lytic potential of viruses to kill tumor cells. Although adenoviruses demonstrate several advantages over other vectors - such as the unparalleled transduction efficacy and natural tropism to a wide range of tissues - the gene transfer efficacy to cancer cells has been limited, consequently restricting the therapeutic effect. There are, however, several approaches to circumvent this problem. We utilized different modified adenoviruses to obtain information on adenovirus tropism towards non-small cell lung cancer (NSCLC) cells. To enhance therapeutic outcome, oncolytic adenoviruses were evaluated. Further, to enhance gene delivery to tumors, we used mesenchymal stem cells (MSCs) as carriers. To improve adenovirus specificity, we investigated whether widely used cyclooxygenase 2 (Cox-2) promoter is induced by adenovirus infection in nontarget cells and whether selectivity can be retained by the 3 untranslated region (UTR) AU-rich elements. In addition, we investigated whether switching adenovirus fiber can retain gene delivery in the presence of neutralizing antibodies. Our results show that adenoviruses, whose capsids were modified with arginine-glycine-aspartatic acid (RGD-4C), the serotype 3 knob, or polylysins displayed enhanced gene transfer into NSCLC cell lines and fresh clinical specimens from patients. The therapeutic efficacy was further improved by using respective oncolytic adenoviruses with isogenic 24bp deletion in the E1A gene. Cox-2 promoter was also shown to be induced in normal and tumor cells following adenovirus infection, but utilization of 3 UTR elements can increase the tumor specificity of the promoter. Further, the results suggested that use of MSCs could enhance the bioavailability and delivery of adenoviruses into human tumors, although cells had no tumor tropism per se. Finally, we demonstrated that changing adenovirus fiber can allow virus to escape from existing neutralizing antibodies when delivered systemically. In conclusion, these results reveal that adenovirus gene transfer and specificity can be increased by using modified adenoviruses and MSCs as carriers, and fiber modifications simultaneously decrease the effect of neutralizing antibodies. This promising data suggest that these approaches could translate into clinical testing in patients with NSCLC refractory to current modalities.

Mutations in beta ` subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

P>Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the beta' subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the beta' subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the beta' subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance.

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.