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PubMed2004

Functional organization of the parabrachial complex and intertrigeminal region in the control of breathing.

Although the medulla oblongata contains the epicenter for respiratory rhythm generation, many other parts of the neuraxis play significant substratal roles in breathing. Accumulating evidence suggests that the pons contains several groups of neurons that may belong to the central respiratory system. This article will review data from microstimulation mapping and tract-tracing studies of the parabrachial complex (PB) and intertrigeminal region (ITR). Chemical activation of neurons in these areas has distinct effects on ventilatory and airway muscle activity. Tract-tracing experiments from functionally identified sites reveal specific respiratory-related sensory inputs and outputs that are likely anatomical substrates for these effects. The data suggest that an important physiological role for the rostral pons may be reflexive respiratory responses to airway stimuli.

Animals↗
PubMed2005

Method for detecting classes of microcystins by combination of protein phosphatase inhibition assay and ELISA: comparison with LC-MS.

Depending on the class of microcystin the protein phosphatase inhibition assay shows different sensitivities to different classes of toxin. We have determined that the IC50 values obtained from dose-response curves for the inhibition of the enzyme by micro-cystin LR, nodularin, YR, and RR were 2.2, 1.8, 9 and 175 nM, respectively. When equimolar amounts of these toxins were determined by the ELISA assay with microcystin LR as the standard, the assay showed equivalence in toxin responses. However, when the toxins were determined by the protein phosphatase inhibition assay using microcystin LR as the standard, the ratios of the values determined by PP-2A to ELISA decreased in the order: nodularin (2.23) microcystin LR (1.1)> microcystin YR (0.63)> microcystin RR (0.06). When the ratios for each standard were plotted against the IC50 values, the log-log plot was negative linear, and the lowest value for the IC50 corresponded with the lowest ratio. The differential sensitivity of the PP-2A assay to the various standards was used to establish an indicative toxicity ranking (ITR) where a ranking of 1 (the highest) was assigned to ratios of > or = 0.8 or greater, and 3 (the lowest) to values < or = 0.2. The three ranking classes corresponded to toxin equivalence represented by the four standards. The new method allows not only the determination of microcystin toxins in terms of stoichiometry (ELISA) but also in terms of indicative toxicity. The method can be performed using the same instrument (e.g. multiwell fluorimeter with absorbance capability) and offers an advantage to methods presently used to determine microcystins (e.g. ELISA or LC-MS). The former has the propensity to overestimate toxicity because it measures equivalence to microcystin LR and is a stoichiometric measurement and the latter has the disadvantage in that relatively few of the microcystins that occur naturally are available as standards. The new method was applied to the analysis of sample from lakes and streams from temperate locations and to extracts of cyanobacterial mats from ponds and streams in cold temperature locations.

Bacterial Toxins↗
PubMed2004

Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors.

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.

Adenoviridae↗
PubMed2005

Complete nucleotide sequence and genomic organization of hepatopancreatic parvovirus (HPV) of Penaeus monodon.

We have determined the genome of hepatopancreatic parvovirus (HPV), a minus, single-stranded DNA virus isolated from infected Penaeus monodon in Thailand. Its genome consisted of 6321 nucleotides, representing three large open reading frames (ORFs) and two non-coding termini. The left (ORF1), mid (ORF2), and right (ORF3) ORFs on the complementary (plus) strand may code for 428, 579, and 818 amino acids, equivalent to 50, 68, and 92 kDa, respectively. The 5' and 3' ends of viral genome contained hairpin-like structure length of approximately 222 and 215 bp, respectively. No inverted terminal repeat (ITR) was detected. The ORF2 contained conserved replication initiator motif, NTP-binding and helicase domain similar to NS-1 of other parvoviruses. Therefore, it most likely encoded the major nonstructural protein (NS-1). The ORF1 encoded putative nonstructural protein-2 (NS-2) with unknown function. The ORF3 of the HPV genome encoded a capsid protein (VP) of approximately 92 kDa. This may be later cleaved after arginine residue to produce a 57-kDa structural protein. A phylogenetic tree based on conserved amino acid sequences (119 aa) revealed that it is closely related to Brevidensoviruses, which are shrimp parvovirus (IHHNV) and mosquito densoviruses (AaeDNV and AalDNV). However, the overall genomic organization and genome size of HPV were different from these parvoviruses, for instance, the non-overlapping of NS1 and NS2, the larger VP gene, and the bigger genome size. This suggested that this HPV virus is a new type in Parvoviridae family. We therefore propose to rename this virus P. monodon densovirus (PmDNV).

Amino Acid Sequence↗
PubMed2006

Adeno-associated virus interactions with B23/Nucleophosmin: identification of sub-nucleolar virion regions.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four replication proteins (Rep78, 68, 52 and 40) encoded by AAV are pleiotropic effectors of virus integration, replication, transcription and virion assembly. Using Rep68 column chromatography and mass spectrometry, we have identified the nucleolar, B23/Nucleophosmin (NPM) protein as an Rep-interacting partner. Rep-NPM interactions were verified by co-immunofluorescence and chemical cross-linking studies. We have found that there is demonstrable, but limited co-localization between Rep and NPM in co-infected cells. In contrast, there was significant co-localization between NPM and AAV Cap proteins. In vitro experiments using purified MBPRep78 and NPM show that NPM stimulates MBPRep78 interactions with the AAV ITR as well as endonuclease activity. These studies suggest that NPM plays a role in AAV amplification affecting Rep function and virion assembly.

Cell Nucleolus↗
PubMed2005

Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors.

Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications.

Animals↗
PubMed2005

Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.

Recombinant adeno-associated virus vectors (rAAV) have been successfully used for long-term gene expression in animal models and in patients. However, while the therapeutic potential of rAAV appears promising, safety issues, including contaminants found in vector stocks, must be further evaluated. We previously reported that a cis-acting replication element present within the AAV-2 p5 promoter was responsible for the encapsidation of rep-cap sequences observed during rAAV production. In that study, we also noticed that plasmid-derived prokaryotic sequences (such as the ampicillin resistance gene) could be found packaged into AAV capsids. In this report, first we confirmed and extended the latter observation by analyzing rAAV stocks produced using different procedures. Second, we demonstrated that these plasmid-derived sequences were transferred and persisted in vivo after rAAV injection into different tissues. Third, our data showed that at least some of these packaged plasmid molecules were linked to the AAV ITRs and were present in vivo in a form that could be rescued through bacterial transformation. This study highlights the need for more stringent characterization of rAAV stocks and provides useful information on the development of rAAV production methods that are able to circumvent or limit the generation of such undesirable particles.

Animals↗
PubMed2005

Identification of novel insertion sites in the Ad5 genome that utilize the Ad splicing machinery for therapeutic gene expression.

Therapeutic transgene expression from oncolytic viruses represents one approach to increasing the effectiveness of these agents as cancer therapeutics. In the case of the oncolytic adenovirus (Ad), however, the genomic packaging capacity is constrained. To address this, we explored whether a transposon-based system could identify sites in the viral genome where endogenous Ad promoters could drive transgene expression via splicing and still maintain the replication capacity of the virus. Using GFP as a reporter gene and an E3-deleted Ad genome as a target, we tested three splicing signals. RACE analysis confirmed that gene expression from the GFP-expressing Ads occurs via splicing and traced expression to the Ad major late promoter (MLP). Replacement of the GFP transposon by an equivalent splice acceptor-luciferase expression cassette in the same orientation confirmed that substitute transgenes are also expressed via splicing from the MLP. Interestingly, insertion of the substitute transgene in the opposite orientation also resulted in expression that, in some cases, originated from within the ITR region of the viral genome. In summary, splice acceptor sequences can be used to control transgene expression from endogenous Ad promoters and this represents a genomically economical approach to arming oncolytic Ads.

Adenoviridae↗
PubMed2004

SHN-1, a Shank homologue in C. elegans, affects defecation rhythm via the inositol-1,4,5-trisphosphate receptor.

Protein localization in the postsynaptic density (PSD) of neurons is mediated by scaffolding proteins such as PSD-95 and Shank, which ensure proper function of receptors at the membrane. The Shank family of scaffolding proteins contain PDZ (PSD-95, Dlg, and ZO-1) domains and have been implicated in the localizations of many receptor proteins including glutamate receptors in mammals. We have identified and characterized shn-1, the only homologue of Shank in Caenorhabditis elegans. The shn-1 gene shows approximately 40% identity over 1000 amino acids to rat Shanks. SHN-1 protein is localized in various tissues including neurons, pharynx and intestine. RNAi suppression of SHN-1 did not cause lethality or developmental abnormality. However, suppression of SHN-1 in the itr-1 (sa73) mutant, which has a defective inositol-1,4,5-trisphosphate (IP(3)) receptor, resulted in animals with altered defecation rhythm. Our data suggest a possible role of SHN-1 in affecting function of IP(3) receptors in C. elegans.

Adaptor Proteins, Signal Transducing↗
PubMed1997

The Deinococcus radiodurans uvr A gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria.

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.

Adenosine Triphosphatases↗
PubMed1997

Molecular evolution of the second ancient human mariner transposon, Hsmar2, illustrates patterns of neutral evolution in the human genome lineage.

A consensus sequence for the second ancient mariner identified in the human genome, Hsmar2, was constructed by majority rule from full-length and partial sequences of 44 of the +/-1000 copies in the genome. This 1300 base pair (bp) consensus has 31 bp imperfect terminal repeats (ITRs) and encodes a 351 amino acid (aa) mariner transposase. The sequence of this transposase has allowed classification of Hsmar2 as a basal lineage of the irritans subfamily of mariners, sharing at most 38% aa identity with other members of the subfamily. The individual copies in the human genome are all highly mutated from the consensus, having suffered numerous small and some large insertions and deletions (indels), including many insertions of S and J subfamily Alu elements. The copies differ, on average, from the consensus by 11.6%, have suffered 11.8 indels per kilobase (kb), and only 3.7% of the 30 hypermutable CpG dinucleotide pairs in the consensus remain intact. This level of divergence indicates that the ancestrally active Hsmar2 element represented by the consensus was present in the human genome lineage about 80 million years (Myr) ago. Each copy has apparently evolved since then largely independently of the others, and with little constraint on its transposase coding capacity. This pattern of molecular evolution fits the current model for mariner transposon evolution. These copies provide multiple independent datasets for evaluating the pattern of neutral evolution in the human genome, for example, they confirm that most indels are very short and that deletions are twice as common as insertions.

Amino Acid Sequence↗
PubMed2001

Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum.

In C. elegans, a hyperactivated MEC-4(d) ion channel induces necrotic-like neuronal death that is distinct from apoptosis. We report that null mutations in calreticulin suppress both mec-4(d)-induced cell death and the necrotic cell death induced by expression of a constitutively activated Galpha(S) subunit. RNAi-mediated knockdown of calnexin, mutations in the ER Ca(2+) release channels unc-68 (ryanodine receptor) or itr-1 (inositol 1,4,5 triphosphate receptor), and pharmacological manipulations that block ER Ca(2+) release also suppress death. Conversely, thapsigargin-induced ER Ca(2+) release can restore mec-4(d)-induced cell death when calreticulin is absent. We conclude that high [Ca(2+)](i) is a requirement for necrosis in C. elegans and suggest that an essential step in the death mechanism is release of ER-based Ca(2+) stores. ER-driven Ca(2+) release has previously been implicated in mammalian necrosis, suggesting necrotic death mechanisms may be conserved.

Amino Acid Sequence↗
PubMed2003

P elements are found in the genomes of nematoceran insects of the genus Anopheles.

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.

Amino Acid Sequence↗
PubMed2006

[Organization and transcription strategy of genome of Bombyx mori bidensovirus (China isolate) VD1].

To better understand the genomic structure and function of Bombyx mori bidensovirus (China isolate) VD1, the VD1 was purified and cloned into the pUC119 vector, and the complete nucleotide sequence of VD1 was determined. Sequence analysis showed that VD1 genome consisted of 6543 nts including inverted terminal repeats (ITRs) of 224 nts. In the viral genome, three major open reading frames (ORF1, ORF2 and ORF3) in the plus strand and one major ORF (ORF4) in the complementary strand were identified. Comparison of the complete genome sequence between Bombyx mori bidensovirus (China isolate) and BmDNV-2 (Yamanashi isolate) showed an identity of 98.4% in VD1, with a total number of 104 bp substitutions and 1 bp insertions found in Bombyx mori bidensovirus (China isolate), the highly variable regions were mainly located in VD1 ORF3 and VD1 ORF4. Northern blotting revealed that VD1 contained 1.1 kb and 1.5 kb transcript in the left-half 'plus' strand, and one transcript about 3.3 kb of 'minus' strand in the right-half. Sequencing of 3' and 5' ends of transcript products showed the 1.1 kb transcript started at nt 290 and ended at nt 1437, the 1.5 kb transcript was found to start nt 1423 and ended at 2931, and the 3.3 kb transcript was found to start nt 6287 and ended at nt 2922. Therefore, the 1.5 kb transcript in the left-half plus' strand and 3.3 kb transcripts of minus' strand in the right-half overlapped for 10 nts at the 3' ends. These results indicate that this virus employs a transcription strategy that is radically different from that of the other reported DNVs.

Amino Acid Sequence↗
PubMed2000

Survey of mariner-like elements in the housefly, Musca domestica.

The presence of mariner-like elements in four strains of the housefly, Musca domestica, was surveyed by PCR. Using the inverted terminal repeat (ITR) sequences of the Mos 1 element as primers, DNAs were successfully amplified from all strains of the housefly. Southern blot analysis indicated that these amplified DNAs were repetitive sequences in the genome of M. domestica. Sequence analyses of cloned PCR products showed that they were 45% identical to the Mos 1 element. These fragments appeared to be nonfunctional, because they contained no intact open reading frame (ORF) capable of encoding transposase. We conclude that these DNAs are degraded mariner-like elements (MLEs) in M. domestica. Because these endogenous MLEs in M. domestica do not encode any functional proteins, they probably would not affect the behavior of mariner-based vectors if such were introduced into this species as transformation vectors.

Animals↗
PubMed1998

Genetic organization and DNA sequence of early region 4 of bovine adenovirus type 3.

We have identified and sequenced 3614 nucleotides located at the extreme right-end of the bovine adenovirus type 3 (BAV3) genome from map units 89.5-100. Analysis of the sequence revealed an inverted terminal repeat (ITR) of 195 bp, and identified five open reading frames (ORFs) designated ORF1, ORF2, ORF3, ORF4 and ORF5. When compared with known E4 ORFs of other adenoviruses, ORFs 1, 2 and 4, which code for proteins of 143, 69 and 143 amino acids respectively, were found to be unique to BAV3. ORFs 3 and 5, which code for proteins of 268 and 219 amino acids respectively, showed partial homology to the E4 34 kDa protein of human adenovirus 2. Nucleotide sequence analysis also identified two potential TATA boxes upstream of ORF1 and a potential polyadenylation signal downstream of ORF5 suggesting that E4 transcripts may be 3' co-terminal.

Adenoviridae↗
PubMed2003

Identification and chromosomal localization of mariner-like elements in the cabbage moth Mamestra brassicae (Lepidoptera).

A complete mariner-like element has been identified in the lepidopteran Mamestra brassicae. This element, called Mbmar, represents a new type of mariner transposon. It has a transposase similar to that of other insect mariner coding sequences but its inverted terminal repeats differ from typical mariner ones. This observation is unique since generally both mariner coding region and ITRs are evolutionarily conserved in insects. Mbmar is detectable by FISH only in the heterochromatic regions of both the sex chromosomes.

Animals↗
PubMed2000

Glucose-responsive gene delivery in pancreatic Islet cells via recombinant adeno-associated viral vectors.

PURPOSE: Recent progress in genetic engineering presents the possibility of providing physiologically regulated glucose metabolism in individuals with diabetes. The objective of this study is to explore the feasibility of obtaining glucose dependent gene expression in the pancreatic beta-cell lines via recombinant adeno-associated virus type 2 (rAAV) mediated gene transfer. METHODS: Two transcription cassettes containing the luciferase gene under the control of the rat insulin I gene promoter and the enhanced green fluorescent protein (EGFP) open reading frame under the control of the immediate early gene promoter of human cytomegalovirus (CMV) were placed in series between the inverted terminal repeats (ITRs) of AAV. The rAAV vectors produced were used to transduce pancreatic beta-cell line grown in the absence or presence of various concentrations of glucose. Luciferase activity assays were performed at 72 hr post-transduction. RESULTS: Glucose-responsive reporter gene expression was obtained in both calcium phosphate transfected HIT-T15 and betaHC-9 cells, demonstrating regulated luciferase gene expression under control of the insulin gene promoter. At MOI of 100, rAAV-transduced betaHC-9 cells exhibited glucose-dependent luciferase activities, which were approximately 4.3 fold higher than those transfected by the calcium phosphate coprecipitation method at 20 mM glucose. CONCLUSIONS: Delivery of the insulin gene promoter via rAAV was shown in this study to result in glucose-dependent control of the reporter gene expression. The results suggest that rAAV is an efficient viral vector for gene transfer into the pancreatic islet cells.

Animals↗