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PubMed2011

Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

Amino Acid Sequence↗

PubMed1996

Theoretical uncertainty of measurements using quantitative polymerase chain reaction.

Current quantitative polymerase chain reaction (PCR) protocols are only indicative of the quantity of a target sequence relative to a standard, because no means of estimating the amplification rate is yet available. The variability of PCR performed on isolated cells has already been reported by several authors, but it could not be extensively studied, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We used the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offspring of a single molecule. We demonstrated that the rate of amplication has a severe influence on the shape of this distribution. For high values of the amplification rate, the distribution has several maxima of probability. A single amplification trajectory is used to estimate the initial copy number of the target sequence as well as its confidence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the early stage of amplification is that small copy numbers result in relatively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements using quantitative PCR. We expect these results to be applicable to the data produced by the next generation of thermocyclers for quantitative applications.

Biometry↗

PubMed1990

Spatial harmonics and pattern specification in early Drosophila development. Part I. Bifurcation sequences and gene expression.

Molecular probes have now provided an unprecedented wealth of detail revealing the changing spatial patterns of gene products in early Drosophila development. This is examined for dynamic properties which might provide insights into the underlying behaviour of the patterning process. What emerges is that transcripts and protein products of members of the major categories of zygotically active genes involved in segmentation pass through transient spatial patterns that are suggestive of harmonic sequences arising from spatial frequency-doubling bifurcations. That is to say, these patterns are typically periodic in space and show a doubling in the number of domains of spatial expression as development proceeds. One of these patterns reflects the primary functional role of the gene in the establishment of the spatial pattern. The different categories of segmentation gene pass through these transients at different rates, those with the longest functional wavelength progressing most slowly. Each gene in a category has its own unique phase relationship to other members, as well as particular variations on the harmonic sequence theme. The result is that the developing embryo experiences a spatial hierarchy of phase-shifted patterning influences that span the range from the whole embryo to single segments, providing progressively more spatial resolution in the patterning process. The characteristic transients and the dynamic relationships between genes of the different categories suggest that gene products expressed in longer-wavelength patterns act as bifurcation parameters on the dynamic system generating the next shorter wavelength category. Such parametric influences are known to result in frequency-doubling bifurcations in Turing reaction-diffusion systems. A general model is proposed of a hierarchically-nested set of quasi-autonomous dynamic systems involving gene activities that can generate the progressively finer spatial order that emerges during embryogenesis. This model has implications for the general stability properties of evolving epigenetic systems.

Animals↗

PubMed2004

Isolation and evaluation of dextral-specific and dextral-enriched cDNA clones as candidates for the handedness-determining gene in a freshwater gastropod, Lymnaea stagnalis.

The handedness of gastropods is genetically determined. The freshwater gastropod Lymnaea stagnalis is a normally dextral species, but contains minor sinistral populations. The gene responsible for handedness determination in this species is predicted to function maternally and specifically in the dextral-ovipositing snail. In this study, we used differential screening and cDNA subtraction to isolate eight "dextral genes" that are specific to, or enriched in, the dextral-ovipositing strains of L. stagnalis. These genes were promising candidates for the handedness-determining gene. In order to determine whether the true handedness-determining gene was among them, we tested for genetic correlations between the level of expression of each dextral gene and the handedness phenotype, i.e., the chirality of the next generation offspring, by using a collection of backcross F2 progeny of F1 offspring from crosses between dextral and sinistral strains. Although the present study could not identify the handedness-determining molecules, this approach appears to be promising for the isolation of such developmentally important genes.

Amino Acid Sequence↗

PubMed2004

Towards a mutant map of the mouse--new models of neurological, behavioural, deafness, bone, renal and blood disorders.

With the completion of the first draft of the human genome sequence, the next major challenge is assigning function to genes. One approach is genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes of interest and subsequent mapping and identification of the mutated genes in question. We (a consortium made up of GlaxoSmithKline, the MRC Mammalian Genetics Unit and Mouse Genome Centre, Harwell, Imperial College, London, and the Royal London Hospital) have used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for use as animal models of human disease and for gene function assignment (Nolan et al., 2000). As of 2003, 35,000 mice have been produced to date in a genome-wide screen for dominant mutations and screened using a variety of screening protocols. Nearly 200 mutants have been confirmed as heritable and added to the mouse mutant catalogue and, overall, we can extrapolate that we have recovered over 700 mutants from the screening programme. For further information on the project and details of the data, see http://www.mgu.har.mrc.ac.uk/mutabase.

Animals↗

PubMed2005

High-throughput magnetic resonance imaging in mice for phenotyping and therapeutic evaluation.

High-throughput mouse magnetic resonance imaging (MRI) is seeing rapidly increasing demand in development of therapeutics. Recent advances including higher-field systems, new gradient and radio frequency coils and new pulse sequences, coupled with efficient animal preparation and data handling, allow high-throughput MRI under certain protocols. However, with current shifts from anatomic to functional and molecular imaging, innovative technology is required to meet new throughput demands. The first multiple mouse imaging strategies have provided a glimpse of the future state-of-the-art. However, the successful translation of standard clinical MRI technology to preclinical MRI is required to facilitate next-generation high-throughput MRI.

Animals↗

PubMed1993

Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

Amino Acid Sequence↗

PubMed2001

ADHD treatment in the 21st century: pushing the envelope.

Required for optimal intervention for attention deficit hyperactivity disorder (ADHD) is evidence-based matching of child, treatment, and situation. The landmark Multimodal Treatment Study (MTA) of Children with ADHD documented the superiority of pharmacotherapy for the vast majority of children with ADHD. Although this study could not address the problem of the match directly, it is generating important leads for research on the use of psychosocial strategies to enhance the scope and durability of treatment gains while decreasing the risks attendant upon long-term use of medication. Given the inherent distinctions between pharmacological and psychosocial treatments, conclusive answers to questions about comparative efficacy will continue to elude scientist-practitioners. Needed next is research examining ways to improve outcomes beyond the effects of medication, using systematically tailored and sequenced psychosocial approaches and exploring new treatment targets, agents, and modalities. To illustrate, some emerging findings from an ongoing experience sampling study and implications for online therapy are discussed.

Attention Deficit Disorder with Hyperactivity↗

PubMed1994

Purification and characterization of UDP-GlcNAc:IV3 beta Gal-Gb4Cer beta-1,6-GlcNAc transferase from mouse kidney.

The Gsl-5 gene mapped on mouse chromosome 19 controls the expression of IV6 beta Gal beta 1-4(Fuc alpha 1-3)GlcNAc, IV3 beta Gal-Gb4Cer in mouse kidney through regulation of the activity of UDP-GlcNAc:IV3 beta Gal-Gb4Cer beta-1,6-Glc-NAc transferase (GNT), which transfers GlcNAc to the C-6 position of GalNAc of IV3 beta Gal-Gb4Cer (GL-X). Here we report that GNT has been purified to apparent homogeneity from mouse kidney by means of preparation of a microsomal fraction, solubilization with Triton X-100, and sequential column chromatographies on CM-Sepharose, UDP-hexanolamine-Sepharose, and Gg4Ose-Aminocellulo-fine. GNT purified 11,000-fold from the microsomal fraction exhibited a specific activity of 26.15 mumol/min/mg protein and an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. The Km values for UDP-GlcNAc and GL-X were 0.36 and 0.11 mM, respectively. Among glycolipids tested as substrates, GL-X was the best and Gg4Cer the next best, but Lc3Cer, Gb4Cer, and GM1 did not act as a substrate. GNT was also able to transfer GlcNAc to Gal beta 1-3GalNAc beta 1-benzyl and Gal beta 1-3GalNAc alpha 1-p-nitrophenyl at C-6 of GalNAc through beta linkage but not to GlcNAc beta-3GalNAc alpha 1-p-nitrophenyl. The purified GNT was digested with lysyl endopeptidase, and four peptides generated were sequenced. The sequence of four peptides spanning 35 amino acid residues in total exhibited 80% homology with that of the reported human core 2 GlcNAc transferase.

Amino Acid Sequence↗

PubMed2001

Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis.

Our previous results have indicated that the major cellular pool of sphingomyelin present on the outer leaflet of the plasma membrane is not involved in the ceramide pathway of apoptosis. Thus, in this study we aimed at defining which intracellular pools of sphingomyelin and ceramide are involved in cell death. The bacterial sphingomyelinase (SMase) gene fused with green fluorescent protein was subcloned into mammalian vectors containing sequences that target the fusion proteins to cytoplasm, plasma membrane, mitochondria, Golgi apparatus, endoplasmic reticulum, or nucleus. Transfection of MCF7 breast cancer cells showed for all constructs an increase in SMase activity ranging from 2- to 60-fold, concomitant with an increase in total cellular ceramide levels (10-100%) as compared with vector-transfected cells. Next, the effect of overexpression of the SMase on cell death was examined. Results demonstrate that only when bacterial SMase was targeted to mitochondria did cells undergo apoptosis; its targeting to the other intracellular compartments was ineffective. Further, the results show that apoptosis induced by mitochondrial targeting of bacterial SMase requires SMase catalytic activity, is prevented by the overexpression of Bcl-2, and is mediated by inducing cytochrome c release. These results demonstrate that ceramide induces cell death specifically when generated in mitochondria. The results highlight the significance of compartment-specific lipid-mediated cell regulation, and they offer a novel general approach for these studies.

Amino Acid Sequence↗

PubMed1991

Reversible inactivation of a transgene in Arabidopsis thaliana.

Fifty percent of Arabidopsis thaliana plants transgenic for a hygromycin resistance gene failed to transmit the resistance phenotype to the progeny. The complete transgene was, however, inherited in all cases according to Mendelian laws as observed by Southern analysis. This discrepancy between genotype and phenotype was the result of a reduced level of transcript in the sensitive transformants. The gene inactivation occurred in plants with multicopy integration of the foreign DNA. No definite correlation was found between gene inactivity and methylation of cytidine residues in the transgene sequence. Explants from several sensitive transformed plants regained a low level of hygromycin resistance on callus induction medium. Subsequent generations obtained by self-pollination were sensitive. In contrast, spontaneous restoration of hygromycin tolerance was observed in seedlings originating from out-crosses with wild-type plants or a different sensitive transformant. A reduction of the copy number was not a prerequisite for spontaneous reactivation. The resistance was often lost again in the next generation. Inactivation and reactivation of the transgene are therefore reversible.

Blotting, Northern↗

PubMed2001

Human DNA polymerase iota promiscuous mismatch extension.

Human DNA polymerase iota is a low-fidelity template copier that preferentially catalyzes the incorporation of the wobble base G, rather than the Watson-Crick base A, opposite template T (Tissier, A., McDonald, J. P., Frank, E. G., and Woodgate, R. (2000) Genes Dev. 14, 1642-1650; Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and Prakash, L. (2000) Nature 406, 1015-1019; Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Mol. Cell. Biol. 20, 7099-7108). Here, we report on its ability to extend all 12 possible mispairs and 4 correct pairs in different sequence contexts. Extension from both matched and mismatched primer termini is generally most efficient and accurate when A is the next template base. In contrast, extension occurs less efficiently and accurately when T is the target template base. A striking exception occurs during extension of a G:T mispair, where the enzyme switches specificity, "preferring" to make a correct A:T base pair immediately downstream from an originally favored G:T mispair. Polymerase iota generates a variety of single and tandem mispairs with high frequency, implying that it may act as a strong mutator when copying undamaged DNA templates in vivo. Even so, its limited ability to catalyze extension from a relatively stable primer/template containing a "buried" mismatch suggests that polymerase iota-catalyzed errors are confined to short template regions.

Base Pair Mismatch↗

PubMed2004

Isolation of nlz2 and characterization of essential domains in Nlz family proteins.

In this study, we first cloned nlz2, a second zebrafish member of the nlz-related zinc-finger gene family. nlz2 was expressed together with nlz1 in a broad posterior domain during gastrula stages as well as at the midbrain-hindbrain boundary and in the hindbrain caudal to rhombomere 4 during segmentation. nlz2 was also expressed in regions distinct from nlz1, notably in the forebrain, midbrain, and trunk. Misexpression of nlz2 in zebrafish embryos disrupted gene expression in the rostral hindbrain, similar to the effect of misexpressing nlz1. We next compared the nlz1 and nlz2 sequences to identify and characterize domains conserved within this family. We found a C-terminal domain required for nuclear localization and two conserved domains (the Sp motif and a putative C(2)H(2) zinc finger) required for nlz1 function. We also demonstrate that Nlz1 self-associated via its C terminus, interacted with Nlz2, and bound to histone deacetylases. Last, we found two forms of Nlz1 generated from alternative translation initiation sites in vivo. These forms have distinct activities, apparently depending on the function of the N-terminal Sp motif. Our data demonstrate that nlz2 functions similarly to nlz1 and define conserved domains essential for nuclear localization, self-association, and corepressor binding in this novel family of zinc-finger genes.

Base Sequence↗

PubMed1995

Methodology of spontaneous baroreflex relationship assessed by surrogate data analysis.

We tested the hypothesis that the spontaneous beat-by-beat interactions of systolic blood pressure (SBP) and R-R interval reflected true baroreflex events rather than chance interactions. Original data sets of 1,024 heartbeats obtained in seated rest from six healthy subjects [R-R interval = 953 +/- 94 (+/- SE) ms] were compared with isospectral [generated by a windowed (inverse) Fourier transform with phase randomization] and isodistribution (data points randomly shuffled) surrogate data sets. The isospectral data set was used to test for random phase relationships, and the isodistribution data set was used for effects of white noise between SBP and R-R interval. Spontaneous baroreflex sequences were defined as three or more beats in which SBP and the R-R interval of the same (lag 0), next (lag 1), or next following (lag 2) beat changed in the same direction. The total number of baroreflex sequences in the original data was significantly greater than the surrogates (P < 0.001). In the original data, there were significantly (P < 0.001) more lag 0 than lag 1 or lag 2 baroreflex sequences. Therefore, these results indicated that spontaneous baroreflex sequences represented physiological rather than chance interactions and that baroreflex responses can occur within the same beat.

Adult↗

PubMed2026

Unlocking the unexplored AMPSphere in marine rare species.

BACKGROUND: Antimicrobial peptides (AMPs) have advantages over traditional antibiotics in fighting against drug-resistant bacterial infections. Natural microbial communities are considered as the priority targets for next-generation AMP bioprospecting initiatives. While progress has been made in characterizing AMPs from the dominant microbial taxa in natural ecosystems, current research largely overlooks the biosynthetic potential of rare species. Given their distinct evolutionary pressures, rare species likely produce AMPs with novel structures and unconventional mechanisms of action. RESULTS: In this study, enrichment cultivation of a marine biofilm was conducted in 138 carbon source- and oxygen level-based conditions, followed by metagenomic sequencing using both Illumina and Nanopore platforms. Analysis of 435 high-quality genomes derived from the metagenomes suggests that these bacterial strains are significantly underrepresented (< 0.01%) in global marine biofilm communities. Through multi-model prediction, we identified 3,054,472 candidate AMPs from the genomes, including 1048 high-confidence ones, thereby significantly expanding the previously known AMPSphere. Furthermore, AMPs derived from the rare bacterial species exhibit unique sequence characteristics, structural diversity, remarkable stability under diverse pH conditions and pepsin exposure, and strong therapeutic potential in animal models, reflecting their specialized adaptive and defensive strategies developed within ecological systems. CONCLUSIONS: The features of the underexplored AMPs from low-abundance bacteria in marine biofilms provide valuable resources and theoretical foundations for the development of highly effective antimicrobial agents. Video Abstract.

Biofilms↗

PubMed2004

Mimicking enzyme evolution by generating new (betaalpha)8-barrels from (betaalpha)4-half-barrels.

Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the (betaalpha)8-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (betaalpha)4-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (betaalpha)8-barrels from (betaalpha)4-half-barrels that were deduced from the enzymes imidazole glycerol phosphate synthase (HisF) and N'[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide isomerase (HisA). To this end, the gene for the C-terminal (betaalpha)4-half-barrel (HisF-C) of HisF was duplicated and fused in tandem to yield HisF-CC, which is more stable than HisF-C. In the next step, by optimizing side-chain interactions within the center of the beta-barrel of HisF-CC, the monomeric and compact (betaalpha)8-barrel protein HisF-C*C was generated. Moreover, the genes for the N- and C-terminal (betaalpha)4-half-barrels of HisF and HisA were fused crosswise to yield the chimeric proteins HisFA and HisAF. Whereas HisFA contains native secondary structure elements but adopts ill-defined association states, the (betaalpha)8-barrel HisAF is a stable and compact monomer that reversibly unfolds with high cooperativity. The results obtained suggest a previously undescribed dimension for the diversification of enzymatic activities: new (betaalpha)8-barrels with novel functions might have evolved by the exchange of (betaalpha)4-half-barrel domains with distinct functional properties.

Aldose-Ketose Isomerases↗

PubMed1992

Independent transposition of multiple Ac elements in the same transgenic tomato cell.

To effectively use transposable elements for the genetic manipulation of plant species lacking well characterized endogenous elements, it is important to evaluate the behavior of known transposable elements following their introduction into heterologous host species. One critical parameter concerns the timing of transposition in relation to the development of the transgenic host since this will affect the frequency with which transposition events are captured in the gametes. In order to examine whether different elements in the same cell are differentially active during development, we used Southern hybridizations to assess the activity of Activator (Ac) elements in progeny plants derived from a tomato transformant carrying five Ac's at two loci. All of the elements at one locus transposed in the primary transformant at a developmental stage resulting in the transmission of newly transposed elements to the next generation. In contrast, one or more of the Ac's at the second locus were not active at this stage and were transmitted to the next generation at the original donor T-DNA insertion site. These elements were, however, transpositionally active in somatic tissue. These results demonstrated that individual transposable elements in the same transformed cell can be differentially activated during development.

Blotting, Southern↗

PubMed2006

Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases.

3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

Binding Sites↗