Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.

Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells.

The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5ß1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

A novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the AAV genome.

Adenovirus and adeno-associated virus (AAV) are eukaryotic DNA viruses being developed as vectors for human gene therapy. The strengths of each system have been exploited in a novel vector that is based on an adenovirus-AAV hybrid virus incorporated into a plasmid-based molecular conjugate. Efficient rescue and replication of the recombinant AAV genome in this hybrid required transient expression of rep. This feature was incorporated into the transducing particle by conjugating a rep expression plasmid to the hybrid virus through a polylysine bridge. The resulting particle is an attractive vehicle for gene therapy because it is easily manufactured and capable of efficiently transducing cells with the end result being rescue and replication of the recombinant AAV genome. This particle is also useful in the production of recombinant AAV resulting in yields 10-fold greater than that achieved with transfection-based protocols.

Deletion of exon 8 causes glycosylasparaginase deficiency in an African American aspartylglucosaminuria (AGU) patient.

We have indentified a GT-to-TT transversion at the splice donor site of intron 8 in the glycosylasparaginase gene from an African American aspartylglucosaminuria (AGU) patient. This mutation causes abnormal splicing of glycosylasparaginase pre-mRNA by joining exon 7 to 9 and excluding 134 bp exon 8. The effect of the mutation is compounded by a frame shift that occurs after the deletion site resulting in premature translational termination. The truncated AGU protein was neither catalytically active nor processed into mature alpha and beta subunits. Both this and a previously characterized Finnish AGU mutation appear to affect folding of the single-chain precursor of glycosylasparaginase and thereby prevent transport of the enzyme to lysosomes.

Genomic structure of human lysosomal glycosylasparaginase.

The gene structure of the human lysosomal enzyme glycosylasparaginase was determined. The gene spans 13 kb and consists of 9 exons. Both 5' and 3' untranslated regions of the gene are uninterrupted by introns. A number of transcriptional elements were identified in the 5' upstream sequence that includes two putative CAAT boxes followed by TATA-like sequences together with two AP-2 binding sites and one for Spl. A 100 bp CpG island and several ETF binding sites were also found. Additional AP-2 and Sp1 binding sites are present in the first intron. Two polyadenylation sites are present and appear to be functional. The major known glycosylasparaginase gene defect G488----C, which causes the lysosomal storage disease aspartylglycosaminuria (AGU) in Finland, is located in exon 4. Exon 5 encodes the post-translational cleavage site for the formation of the mature alpha/beta subunits of the enzyme as well as a recently proposed active site threonine, Thr206.

Cloning and sequence analysis of a cDNA for human glycosylasparaginase. A single gene encodes the subunits of this lysosomal amidase.

We have isolated a full-length cDNA (HPAsn.6) for human placenta glycosylasparaginase using a 221-bp PCR amplified fragment containing rat liver asparaginase gene sequences. The deduced amino acid sequence from the human clone showed sequence identity to both the alpha and beta subunits of the rat enzyme. The human enzyme is encoded as a 34.6 kDa polypeptide that is post-translationally processed to generate two subunits of approx. 19.5 (alpha) and 15 (beta) kDa. A charge enriched region is present at the predicted site where cleavage occurs. Using polyclonal antibodies against the alpha and beta subunits of rat liver asparaginase, we have shown that the human enzyme is similar in structure to the rat enzyme.

Post-translational processing and Thr-206 are required for glycosylasparaginase activity.

Lysosomal glycosylasparaginase is encoded as a 36.5 kDa polypeptide that is post-translationally processed to subunits of 19.5 kDa (heavy) and 15 kDa (light). Recombinant glycosylasparaginase has been expressed in Spodoptera frugiperda insect cells enabling the precursor and processed forms to be isolated and their catalytic potential determined. Only the subunit conformation was functional indicating glycosylasparaginase is encoded as an inactive zymogen. The newly created amino terminal residue of the light subunit following maturation, Thr-206, is believed to be involved in the catalytic mechanism [1992, J. Biol. Chem. 267, 6855-6858]. Here we have constructed two amino acid substitution mutants replacing Thr-206 with Ala-206 or Ser-206 and demonstrate that both destroy enzyme activity.

Physical properties and use of pertechnegas as a ventilation agent.

UNLABELLED: Pertechnegas, a variant of technegas, produces similar ventilation images with a much increased clearance rate. This work aims to determine the properties of pertechnegas and its use as a ventilatory agent. METHODS: Fourteen men and 11 women were scanned for PE, after pertechnegas ventilation. Six were reimaged with technegas within 1 wk. Studies were reported according to PIOPED criteria. Pertechnegas samples were analyzed by transmission electron microscopy (TEM), cascade impaction (CI), aerosol mobility analysis (AMA), Fourier transform mass spectrometry (FTMS), x-ray photoelectron spectroscopy (XPS), paper strip (PC) and gas chromatography (GC). RESULTS: Post-test probabilities were normal in 5, low in 8, high in 5 and indeterminate in 7. There were 15 Grade 1, 6 Grade 2 and 4 Grade 3 studies. All Grade 3 patients had FEV1 < 1.5 liters, 3 with rates < 1.0 liter. Patients with high probability had proven deep venous thrombosis in three by venography and in one by doppler. TEM identified 0.3 micron salt particles. CI demonstrated a 7-min time to half clearance from the chamber for particles in the < 0.1 micron range. AMA indicated all particles were < 0.032 micron when salt was excluded. Pertechnegas behaves in PC as pertechnetate, GC demonstrated CO levels below 516 ppm. CO2 concentrations were 0.146 +/- 0.0009%. FTMS found molecular pertechnetate species including 99TcO3(OH)+, Na99TcO3(OH)3+ and Na99TcO3(OH)3+. XPS confirmed that these Tc species exist in oxidation state +7. CONCLUSION: Comparison with technegas images in the follow-up group proved equivalent in the first five views, but indistinct lung boundaries and a high background activity characterized the final anterior images. The active component of pertechnegas is molecular pertechnetate.

Structural and functional heterogeneity of integrated recombinant AAV genomes.

Adeno-associated Virus (AAV) has emerged as a promising vector for gene therapy because of its ability to generate high titer recombinant stocks and the potential for site specific integration. However, much of the current knowledge regarding the transduction and integration biology of this virus is based on studies evaluating wild type AAV or recombinant AAV which was unknowingly contaminated with wild type virus. Given the fact that recombinant AAV is replication incompetent, by virtue of deleted viral rep proteins responsible for site specific integration of the wild type virus, the integration process for recombinant AAV may likely be different from its wild type counterpart. To this end, the present study has attempted to elucidate the proviral structure of stably integrated recombinant AAV genomes harboring the alkaline phosphatase reporter gene in 293 and IB3 cell lines. Initial studies attempted to functionally characterize differences in proviral genomes using mobilization assays with assessed both liberated episomal recombinant AAV and infectious virus following transfection with Rep/Cap containing plasmids and/or infection with recombinant adenovirus (Ad). Using Southern and polymerase chain reaction (PCR) analysis, evaluation of genomic DNA from AAV clonal cell lines indicated that head to tail orientations of ITRs were absolutely required for excision of episomal genomes and rescue of infectious recombinant virus. Furthermore, mobilization of proviral DNA could be achieved in the presence of exogenous Rep/Cap without adenovirus, while mobilization of infectious recombinant virus required the addition of both Rep/Cap and Ad. Genomic Southerns suggest that two predominant proviral structures exist for recombinant AAV including head to head and tail to head duplex genomes. A third class of monomer proviral genomes with head to tail oriented ITRs was also observed. No evidence for tail to tail ITR oriented proviral genomes was detected in any of the clonal cell lines. Such findings have begun to lay the foundation for a clearer understanding of the mechanism of recombinant AAV integration and how this process differs from wild type AAV.

Sub-ppt detection limits for copper ions with Gly-Gly-His modified electrodes.

An electrochemical metal ion sensor has been developed with a detection limit of less than 0.2 ppt by the covalent attachment of the tripeptide Gly-Gly-His as a recognition element to a 3-mercaptopropionic acid modified gold electrode.

Beliefs about smoking cessation among out-of-school youth.

Although the majority of adolescents in the 13-18 age range are at school, there is a need to target specific groups of young smokers such as unemployed youth. For those young people who are not at school, few directed programs are available in either prevention or cessation and information is needed about the design and delivery of appropriate programs for this population. This report presents the results from a survey of unemployed youth and students at vocational colleges about various aspects of smoking cessation. The majority of out-of-school youth smokers had not tried to quit, but 52% were contemplating action to quit. Only a quarter of the smokers had quit for more than a week. Few young smokers would use a recognised program though more females would change to a lower nicotine brand, quit with the help of a friend or participate in a group quit program. The method of quitting most would recommend to peers is 'use of will power'. Incentives to quit were attractive to only a third of the smokers, and many enhancing and inhibiting factors for participation in programs were identified. In particular, efforts to quit increased their confidence in quitting, supporting the need to assist those who are contemplating action to quit. Programs need to incorporate input from youth and be tailored for them but not necessarily for different groups such as non-secondary school students and unemployed youth.

Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV.

Recombinant adeno-associated virus (rAAV) is a promising vector for retinal application as it transduces photoreceptors and retinal pigment epithelium cells efficiently and in a stable fashion. Because rAAV also transduces retinal ganglion cells, we reasoned that ocular application of rAAV might result in delivery of transgenic protein to the CNS. Here we describe high levels of green fluorescent protein (GFP) persisting at least 6 months in optic nerves and brains of mice and dogs after intravitreal delivery of rAAV-GFP. There was no clinical or histological evidence of inflammatory response although a mild humoral Th-2 response to viral capsid proteins was detected. These findings have important implications with respect to therapeutic applications of rAAV.

The effectiveness of workplace smoking cessation programmes: a meta-analysis of recent studies.

OBJECTIVE: Using meta-analytic procedures, we compare the effectiveness of recent controlled trials of worksite smoking cessation during the 1990s with a previous meta-analysis of programmes conducted in the 1980s. DATA SOURCES: ABI/Inform, BRS, CHID, Dissertation Abstracts International, ERIC, Medline, Occupational Health and Safety Database, PsycInfo, Smoking and Health Database, SSCI, and Sociological Abstracts. STUDY SELECTION: Controlled smoking cessation interventions at the workplace with at least six months follow up published from 1989 to 2001 and reporting quit rates (QRs). DATA EXTRACTION: Two reviewers independently scanned titles/abstracts of relevant reports, and we reached consensus regarding inclusion/exclusion of the full text reports by negotiation. A third reviewer resolved disagreements. Two reviewers extracted data according to a coding manual. Consensus was again reached through negotiation and the use of a third reviewer. DATA SYNTHESIS: 19 journal articles were found reporting studies conforming to the study's inclusion criteria. Interventions included self help manuals, physician advice, health education, cessation groups, incentives, and competitions. A total of 4960 control subjects were compared with 4618 intervention subjects. The adjusted random effects odds ratio was 2.03 (95% confidence interval 1.42 to 2.90) at six months follow up, 1.56 (95% CI 1.17 to 2.07) at 12 months, and 1.33 (95% CI 0.95 to 1.87) at more than 12 months follow up. Funnel plots were consistent with strong publication bias at the first two follow ups but not the third. In Fisher et al's 1990 study, the corresponding ORs were 1.18, 1.66, and 1.18. CONCLUSIONS: Smoking cessation interventions at the worksite showed initial effectiveness, but the effect seemed to decrease over time and was not present beyond 12 months. Compared to the Fisher (1990) analysis, the effectiveness was higher for the six month follow up. Disappointingly, we found methodological inadequacies and insufficient reporting of key variables that were similar to those found in the earlier meta-analysis. This prevented us from determining much about the most effective components of interventions. It is advisable for researchers conducting studies in the future to report data on attrition and retention rates of participants who quit, because these variables can affect QRs.

Healthy behaviors, lifestyle, and reasons for quitting smoking among out-of-school youth.

STUDY OBJECTIVE: To investigate the relationships among healthy behavior, healthy values, social influences, and quitting smoking in adolescents not attending school. DESIGN: Following screening procedures, young smokers independently completed a self-report questionnaire administered by trained staff. SETTING: Vocational (TAFE) colleges and Commonwealth Employment Offices (CES) from varying socioeconomic localities were selected as sites to intercept smoking adolescents on their attitudes about quitting smoking. SUBJECTS: Youth attending vocational colleges or CES. RESULTS AND CONCLUSIONS: There were no differences between the two groups of smokers (vocational students and unemployed youth). The decision to quit smoking among these youth is based on a number of factors including social and personal reasons. Health-oriented values were found to be more highly associated with quitting behavior than social influences. Programs for smoking cessation need to be focused on an overall health and improvement approach rather than only a quit-smoking approach.

The observation of fullerenes in a Technegas lung ventilation unit.

Evidence is provided to show that Technegas has structure compatible with the Buckminsterfullerene model C60 in which 99Tcm atoms are trapped.

Quantification of left ventricular diastolic wall motion by Doppler tissue imaging in healthy cats and cats with cardiomyopathy.

OBJECTIVE: To assess Doppler tissue imaging (DTI) for evaluating left ventricular diastolic wall motion in healthy cats and cats with cardiomyopathy. ANIMALS: 20 healthy cats, 9 cats with hypertrophic cardiomyopathy (HCM), and 9 cats with unclassified cardiomyopathy (UCM). PROCEDURE: A pulsed wave DTI sample gate was positioned at a subendocardial region of the left ventricular free wall in the short axis view and at the lateral mitral annulus in the apical 4-chamber view. Indices of diastolic wall motion were measured, including peak diastolic velocity (PDV), mean rate of acceleration and deceleration of the maximal diastolic waveform (MDWaccel and MDWdecel, respectively), and isovolumetric relaxation time (IVRT). RESULTS: The PDV of cats with HCM and 6 of 9 cats with UCM was significantly decreased, compared with that of healthy cats. In the 3 cats with UCM that had a PDV that was not different from healthy cats, MDWaccel and MDWdecel were greater, and IVRT was shorter than those of healthy cats. The IVRT in cats with HCM was longer than that of other cats. CONCLUSIONS AND CLINICAL RELEVANCE: Indices of diastolic function in cats with HCM, and in many cats with UCM, differed from those of healthy cats and were similar to those reported in humans with HCM and restrictive cardiomyopathy, respectively. However, the hemodynamic abnormality was not the same for all cats with UCM; some cats with an enlarged left atrium and a normal left ventricle (ie, UCM) had abnormal left ventricular wall motion consistent with restrictive cardiomyopathy while others did not.

Strengthening health promotion in Australian workplaces.

The Australian workplace has emerged as an important venue for influencing the health of employees through regulations and behaviour change programs. Recent surveys have highlighted a growth in this activity but the effectiveness of these programs in changing unhealthy work practices and policies is questionable. The need for strengthening programs by stronger designs and evaluation, and addressing organisational factors and employee participation in planning and implementation processes is documented. Efforts in that direction in Queensland are cited, Building on these existing foundations, redirecting existing resources, and building intersectoral cooperation in public-private partnerships hold a creative, exemplary vision of the future for Australian workplace programming.

Patient reports of health education activities in a public hospital.

Hospitals have been greatly underutilised as settings for health promotion activities in Australia. Most research on this subject has been derived from hospital administrators or clinicians. An exit survey of inpatients (n = 460) of a large regional public hospital in Queensland was conducted to examine reported levels of health education in relation to cancer prevention and control. Patients indicated considerable receptivity to a range of secondary prevention activities in the hospital setting, indicating the potential for an increased contribution to the national health agenda by many hospitals.