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At least 55 records · Page 3Linked to original sources
PubMed2004

Effects of enhanced sanctions for high-BAC DWI offenders on case dispositions and rates of recidivism.

Implemented January 1, 1998, Minnesota's high-BAC law mandates more severe administrative pre-conviction penalties and more severe post-conviction penalties for offenses with BACs > or = 0.20%. Most notably, the law provides for the administrative impoundment of the license plate of first-time DWI offenders with BACs > or = 0.20. During the three years after the law took effect, a large majority of first-time and repeat offenders with BACs > or = 0.20% did, in fact, receive high-BAC administrative dispositions and/or high-BAC court convictions, which carried more severe penalties. For example, in 1998 85.6% of first offenders with BACs > or = 0.20% received a high-BAC administrative disposition and/or a high-BAC court conviction; 65.0% received both high-BAC administrative and high-BAC court dispositions. The proportion of high-BAC first-time offenders who received the statutory high-BAC dispositions declined from 1998 to 1999 and 2000. Based on survival analysis, the one-year recidivism rate among first offenders arrested in 1998 with BACs > or = 0.20% was significantly lower than for offenders with BACs 0.17-0.19% (who also had relatively high BACs but were not subject to enhanced sanctions), after controlling for age and gender. There were similar, but not significant, results for first offenders arrested in 1999.

Alcohol Drinking↗
PubMed2006

Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases.

BACKGROUND: Oncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. RESULTS: We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i) two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii) the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV) efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39 mutant). CONCLUSION: Our Flip-Flop HSV-BAC system enables rapid generation of HSV vectors carrying transgene inserts. By introducing a tumor-specific-promoter-driven ICP4 cassette into pM24-BAC using this system, one should be able to generate transcriptionally-targeted oncolytic HSV vectors. We believe this system will greatly facilitate the screening of a plethora of clinically useful tumor-specific promoters in the context of oncolytic HSV vectors.

Chromosomes, Artificial, Bacterial↗
PubMed2000

A gene-enriched BAC library for cloning large allele-specific fragments from maize: isolation of a 240-kb contig of the bronze region.

A generic bacterial artificial chromosome (BAC) library from a complex plant genome like maize may not be suitable for some types of genomic analysis, for example, for establishing correlations between the genetic and the physical organization of a given chromosome region. Previously, we carried out extensive genetic analysis of the bronze (Bz) region in Zea mays using a W22 inbred line carrying the Bz-McC allele; however, BAC libraries of that line are neither available nor under construction. Here, we report the isolation of large, adjacent BAC clones of this region from a partial BAC library of W22. We developed a BAC vector suitable for cloning NotI fragments and used it to clone size-fractionated genomic DNA that had been cut to completion with the methylation-sensitive, rare-cutting enzyme NotI. This strategy resulted in a very significant enrichment of large genic DNA. From a library of about 20,000 BACs, containing just two-thirds of a maize genome, we isolated 16 BAC clones of the 110-kb distal Bz fragment and 10 BAC clones of the 130-kb proximal Bz fragment. This recovery means that our strategy resulted in a 15- to 24-fold enrichment of specific sequences. The order of the BAC clones in the 240-kb contig, predetermined from an internal NotI site in the Bz-McC allele was confirmed by hybridization with sequences from sites previously mapped proximal and distal to Bz and by sequencing. To show the general utility of our approach and the value of our partial BAC library, we also isolated BAC clones of other sequences, such as tub4 and the complex R-r allele, contained in the same size fraction of DNA. This is the first report of the use of a BAC vector to clone allele-specific large DNA fragments from a plant with a large genome, circumventing the need to construct a complete BAC library.

Alleles↗
PubMed2006

Streptococcus agalactiae Cbeta protein gene (bac) sequence types, based on the repeated region of the cell-wall-spanning domain: relationship to virulence and a proposed standardized nomenclature.

The Cbeta protein (Bac) of Streptococcus agalactiae (group B streptococcus; GBS) is an IgA binding protein encoded by bac, of which at least 39 sequence types have been described, based on polymorphisms in the repeated region of the cell-wall-spanning domain ('bac sequence types'). Cbeta is usually found in serotype Ib, less commonly in serotype II, and rarely in other serotypes. The aim of this study was to examine the prevalence, variety and distribution, among GBS serotypes and between invasive and superficial isolates, of bac sequence types. A total of 1101 GBS isolates were tested, from 10 countries, with a bac-specific PCR, and amplicons from all 255 (23 %) with positive results were sequenced. Ninety-seven percent (184/190) of serotype Ib and 37 % of serotype II isolates were bac positive. The Calpha protein gene (bca) was present in 98 % (251/255), and insertion sequences IS1381 and IS861 in 94 % (239/255), of bac-positive isolates. The authors identified 59 bac sequence types belonging to 19 groups, based on length, from 496 to 946 bp, with up to six sequence variants (a-f) in each group. The median bac sequence length of invasive isolates was significantly shorter than that of superficial isolates overall (640 versus 586 bp; P < 0.001) and specifically for serotype Ib (541 versus 676 bp; P < 0.001), and invasive isolates were significantly (P < 0.001) more likely to have one or more 18 bp deletions relative to the original published bac sequence (X59771). bac sequence typing is a useful addition to the previously described genotyping system, and will help to predict relative virulence among S. agalactiae serotype Ib strains.

Antigens, Bacterial↗
PubMed2000

Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization.

We have used sequence-based markers from an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for overgo probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in three stages: (1) superpool hybridizations against the 12x coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12x BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo-BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.

Chromosomes, Bacterial↗
PubMed2004

Corneal epithelial cellular dysfunction from benzalkonium chloride (BAC) in vitro.

PURPOSE: To investigate the functional and morphological toxicity of benzalkonium chloride (BAC) on corneal epithelial cells in vitro. METHODS: Primary corneal epithelial cells were cultured from rabbit cornea. Corneal epithelial cells containing radioactive 51Cr were exposed for 5 min, 10 min, 30 min and 60 min to concentration of BAC 0.001%, 0.005%, 0.01%, 0.05% and 0.1%. Control cells were treated with phosphate buffer solution alone. 51Cr release from epithelial cells into the supernatant was used as an index of epithelial cell lysis. Cell detachment (index of cell dysfunction) was analysed by measuring 51Cr activity in the supernatant and wash fluid. Morphological cell damage was investigated with transmission electron microscopy. RESULTS: With the higher concentration and the longer duration of BAC exposure, corneal epithelial cell lysis was increased significantly (P < 0.05). Cells showed severe damage at BAC concentration over 0.05% during 5 min of exposure. Cell dysfunction appeared markedly at BAC concentrations of 0.005% for 30 min of exposure, but decreased with longer exposure times. There was an increase in significant cytoplasmic damage with longer BAC exposure times, although not with a minimal dose of 0.001%. Disrupted cytoplasmic membranes of corneal epithelial cells appeared at the higher BAC concentration of 0.1%, and at the longer exposure time of 30 min with BAC concentration of at least 0.001%. CONCLUSIONS: BAC can induce corneal epithelial dysfunction, which can damage the corneal epithelial barrier. This effect occurs when BAC is used frequently or for periods over 30 min, even when the BAC concentration is low (0.001%).

Animals↗
PubMed2004

Construction of recombinant polypeptides based on beta antigen C (Bac) protein & their usage for protection against group B streptococcal infection.

BACKGROUND & OBJECTIVES: immunocompromized adults. Approximately 50 per cent of the GBS strains carry and express the gene of BAC antigen which is capable to bind IgA. Gene encoding for the BAC antigen has been cloned and sequenced but actual IgA binding region on the protein has not been detected. The aim of the present work was to localize the region of IgA binding on Bac protein, to evaluate the role of one of the Bac protein regions MLKKIE in IgA binding, and to investigate the ability of Bac based recombinant proteins to generate protective antibodies against GBS infection. METHODS: Recombinant proteins based on beta antigen C were generated after PCR amplification of the fractions of bac gene with the following cloning of the PCR products into expression plasmids. Recombinant peptides were tested for IgA binding by immunoprecipitation and Western blot. One of the recombinant proteins expressing IgA binding was used as an antigen for immunization of mice and for GBS protection studies. RESULTS: Several bac gene constructs were generated. Their ability to bind IgA varied dramatically depending on the size of the construct and location of the fragment on the bac gene map. The smallest peptide expressing IgA binding was 14 kD in size. Amino acid substitutions in MLKKIE region facilitated IgA binding ability. Immunization of mice with recombinant Bac based peptide induced the appearance of anti-GBS antibody with high affinity level providing protection against GBS infection. INTERPRETATION & CONCLUSION: Size dependence of Bac based recombinant peptides proved that the effective IgA binding required specific folding of the protein binding IgA. Region MLKKIE could not be considered as region, responsible for IgA binding. Generation of antibodies against Bac based recombinant peptides with high titre and affinity makes these proteins a potent candidates for generating a vaccine against GBS infection.

Adult↗
PubMed1986

Identification of an early activation antigen (Bac-1) on human B cells.

We have produced a monoclonal antibody, Bac-1, that appears to identify a novel antigen on activated human B cells. The Bac-1 antigen can be detected between 8 to 16 hr, as well as transferrin receptors (T9), after activation of small resting B cells with phorbol myristic acetate, anti-IgM antibody, Staphylococcus aureus Cowan I, or Epstein-Barr virus. The expression of the Bac-1 antigen precedes that of IL 2 receptors (Tac-1). Peak expression of the Bac-1 antigen was observed on day 3 after activation, and decreased thereafter. The Bac-1 antigen was present on a minor subpopulation of relatively large B cells isolated from blood samples, and on "preactivated" B cells of heterogeneous size isolated from spleens and tonsils. It was not detected on bone marrow pre-B cells, blood small B cells, or plasma cells, nor was it expressed by resting or activated T cells or nonlymphoid cells. Certain B cell neoplasms and B lymphoblastoid cell lines were Bac-1+, but neoplastic cells of non-B lineage were Bac-1-. With immunoperoxidase staining, Bac-1+ cells were detected predominantly in the germinal centers of tonsil sections. The Bac-1 antigen on activated B cells was destroyed by protease treatment and was enhanced by neuraminidase treatment, suggesting that the Bac-1 antibody detects a cell surface molecule via an antigenic determinant which is partially obscured by neighboring sialic acid residues. The reactivity pattern of Bac-1 differs from the patterns of cellular reactivity reported for other monoclonal antibodies with specificity for activated human B cells.

Antibodies, Monoclonal↗
PubMed2004

In vitro and in vivo delivery of intact BAC DNA -- comparison of different methods.

BACKGROUND: The ability to deliver large (>100 kb) fragments of DNA to mammalian cells in vitro and in vivo is becoming increasingly important with the availability of BAC and PAC constructs for gene expression. Here we investigate in vitro and in vivo delivery of BACs up to 157 kb. METHODS: Different types of polyethylenimine (PEI) and Lipofectamine were used to deliver 150-kb BAC (bacterial artificial chromosome) DNA to mouse and human cell lines in tissue culture and the level of EGFP expression compared. To assess the intactness of the DNA delivered, a BAC carrying oriP/EBNA-1 was used to make stably transfected cell lines. Episomal DNA was then rescued into E. coli followed by analysis on a pulsed-field gel. Three different methods of in vivo delivery were also assessed for delivery of BAC DNA; intravenous injection of DNA/PEI particles, intramuscular injection with electroporation and high-volume injection into the tail vein. RESULTS: PEI22 (linear polymer form, 22 kDa) was found to be the most efficient method for delivery of 150-kb BAC DNA to both cell lines in tissue culture. However, Lipofectamine 2000 was found to give a higher proportion of intact DNA than PEI22 in stably transformed colonies and almost all the DNA delivered by Lipofectamine 2000 was intact. Intravenous injection of DNA/PEI particles was found to be inefficient for delivery of BAC DNA. Intramuscular injection with electroporation of pure BAC DNA was very efficient and expression was maintained for 105 days. High-volume injection of BAC DNA gave excellent expression in the liver and intact BAC DNA could be rescued 7 days after injection. CONCLUSIONS: These results demonstrate efficient delivery of intact, large (up to 157 kb) DNA constructs for in vitro gene expression and in vivo gene therapy applications.

Animals↗