A multi-level analysis showing associations between school neighborhood and child body mass index.

OBJECTIVE: The objective of this study is to examine associations between aspects of the environment in school neighborhoods and childhood body mass index percentile (BMIp). METHODS: Trained medical students visited 46 elementary schools in the Kansas City metropolitan area to conduct medical screenings that included the height and weight measurements of 12 118 boys and girls 4-12 years of age in the academic year 2008-2009. For the same time period, aspects of the built environment in a 2-mile radius around each school was obtained from the Walkscore database. Other environmental characteristics (for example, population change) of these areas were also obtained from various sources. Hierarchical linear modeling was used to estimate the associations between neighborhood- and individual-level factors and BMIp. RESULTS: Population size along with the number of fast-food restaurants and grocery stores were positively associated with BMIp, whereas population change along with the number of parks and fitness centers were inversely associated with BMIp. CONCLUSIONS: After considering individual-level factors and the random effects of schools, environmental elements of school neighborhoods predict childhood BMIp. This study offers evidence of the health influence of school neighborhoods in a way that can inform neighborhood redevelopment efforts.

Genotypic characterization of Ureaplasma species by pulsed field gel electrophoresis.

We describe the first use of pulsed field gel electrophoresis to genotype human Ureaplasma species. This technique can distinguish between U. urealyticum and U. parvum, differentiate most of the 14 serovars from one another, and identify differences among clinical isolates of the same serovar.

Complete nucleotide sequence and analysis of pPSR1 (72,601 bp), a pPT23A-family plasmid from Pseudomonas syringae pv. syringae A2.

Plasmid pPSR1 is a conjugative plasmid originally isolated from Pseudomonas syringae pv. syringae A2, and is a member of the recently described pPT23A plasmid family. We have determined the complete sequence of pPSR1 and found the plasmid to be 72,601 bp in length, encoding 55 ORFs. Putative functions were assigned to 49 ORFs; of these, 24 (49.0%) are involved in plasmid replication, maintenance or conjugation, 17 (34.7%) have roles in virulence or ecological fitness, and eight (16.3%) encode transposase functions as part of mobile elements. pPSR1 carries the effector gene orf34, the mutagenic DNA repair operon rulAB which confers tolerance to ultraviolet radiation, and two genes for methyl-accepting chemotaxis proteins, one of which was located within the novel transposon Tn 5395. The streptomycin resistance transposon Tn 5393a, which carries a strA-strB determinant, was found inserted immediately downstream of the pPSR1 repA gene. Functional analysis of the replication region of pPSR1 indicated that the repA gene and flanking upstream and downstream sequences are required for autonomous replication in P. syringae. Hybridization analyses of the distribution of 11 of the pPSR1 ORFs indicated that many of the ecologically important ORFs were confined to the pathovar P. syringae pv. syringae -either to strains from the local population from which pPSR1 was originally isolated, or strains from a worldwide collection. Conjugative transfer genes and a gene encoding a transcriptional regulator were more widely distributed among several P. syringae pathovars. The sequence analysis of pPSR1 suggests that pPT23A-family plasmids evolve by accumulating genes that are important for host-pathogen interactions or growth on plant hosts, which are incorporated onto a conserved backbone encoding conjugation and stability determinants.

Endotoxin inactivation in water by using medium-pressure UV lamps.

Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be approximately 0.55 endotoxin unit/ml per mJ/cm(2) of irradiation delivered.

Psoralen-modified clamp-forming antisense oligonucleotides reduce cellular c-Myc protein expression and B16-F0 proliferation.

The c-myc protooncogene plays an important role in the abnormal growth pattern of melanoma cells. In an attempt to inhibit c-Myc expression and the growth of an established murine melanoma cell line, we targeted homopurine sequences within the mouse myc mRNA with modified antisense oligonucleotides (AS ODNs). Psoralen was conjugated to the 5'-end of these clamp-forming oligonucleotides (clamp ODNs). Gel mobility shift analysis demonstrated a sequence-specific interaction between the active clamp ODNs (Myc-E2C and Myc-E3C) and the 1.4 kb c-myc mRNA, but no interaction with the control clamp ODN (SCR**). This association was further confirmed by thermal denaturation studies. In vitro translation assays demonstrated that both Myc-E2C and Myc-E3C at 5 microM inhibited c-Myc expression >99% after UV activation at 366 nm. Immunostaining of B16-F0 cells with a c-Myc monoclonal antibody revealed a significant reduction in c-Myc after clamp ODN treatment compared with the untreated or SCR** control-treated cells. This result was corroborated by western blot analysis. Utilizing the MTT assay to determine the effects of ODN-mediated c-Myc reduction on B16-F0 growth, we observed 60 and 64% reductions in growth after treatment with 5 microM Myc-E3C and Myc-E2C, respectively. We attribute the enhanced effectiveness of the clamp ODNs to psoralen activation. Our preliminary data suggest that inhibiting c-Myc overexpression results in a significant reduction in abnormal proliferation of B16-F0 melanoma cells and that the increased efficiency of clamp ODNs may provide an important advantage for their use in antisense therapies.

A treatment algorithm for pneumothoraces complicating central venous catheter insertion.

BACKGROUND: We investigated the role of observation or insertion of a small French pigtail catheter with Heimlich valve as alternative management to a tube thoracostomy for iatrogenic pneumothorax complicating central venous catheter (CVC) insertion. METHODS: A retrospective review of 9,637 consecutive patients who had had subclavian CVCs inserted on an outpatient basis identified 100 patients with pneumothoraces. Treatment consisted of (1) observation, (2) outpatient insertion of a Heimlich valve, or (3) inpatient tube thoracostomy. RESULTS: The median pneumothorax size was 10% (range 1% to 100%). Fifty-eight patients had observation as initial treatment, and this strategy was successful in 35 (60%). Thirty-four patients were treated initially with Heimlich valves, and this strategy was successful in 29 (85%). Tube thoracostomy as initial therapy was successful in 7 (88%) of 8 patients. Patients in who initial treatment failed were treated with insertion of a Heimlich valve or tube thoracostomy. CONCLUSION: In appropriately selected patients, pneumothorax after insertion of a subclavian CVC can be successfully managed in the outpatient setting with observation. Patients in whom observation fails can be treated with insertion of a Heimlich valve. Tube thoracostomy can be reserved for refractory PTX or emergent situations.

The integral divalent cation within the intermolecular purine*purine. pyrimidine structure: a variable determinant of the potential for and characteristics of the triple helical association.

In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.

Inhibition of transcription of the human c-myc protooncogene by intermolecular triplex.

Triplex-forming oligonucleotides (TFOs) have been shown to inhibit both transcription in vitro and the expression of target genes in cell culture by binding to polypurine/polypyrimidine sequences in several human gene promoters. The c-myc protooncogene is overexpressed in a variety of human cancers and appears to play an important role in the proliferation of these cells. In an attempt to assay the ability of triplex-forming oligonucleotides to inhibit expression of a target gene in vivo, we have developed a cellular system involving transfection of a c-myc promoter-driven luciferase reporter plasmid with triplex-forming oligonucleotides targeted to the human c-myc protooncogene. To increase the stability of the TFO, we have used modified phosphorothioate oligonucleotides. Triplex formation with a modified phosphorothioate oligonucleotide occurs with approximately equal binding affinity as that seen using a phosphodiester oligonucleotide. Phosphorothioate-modified TFOs targeted to c-myc inhibit transcription of the c-myc promoter in HeLa cells as demonstrated by a decrease in luciferase expression from a luciferase reporter gene construct. These results suggests that triplex formation may represent a gene-specific means of inhibiting specific protooncogene expression.

Efficient delivery of triplex forming oligonucleotides to tumor cells by adenovirus-polylysine complexes.

Oligonucleotides (ODNs) show great promise in their ability to specifically inhibit single gene expression but must cross the cell membrane, escape the endosomal vesicle, and possibly traverse the nuclear membrane to arrive at their intracellular target molecules. In an attempt to improve the delivery of phosphodiester triplex forming ODNs to malignant cells, we have constructed adenovirus-polylysine (AdpL)-ODN complexes designed to take advantage of the receptor mediated endocytosis of adenoviruses to transfer the ODNs to the cell nucleus. Treatment of several different types of tumor cells in culture by AdpL-ODN complex resulted in superior uptake and persistence of the ODNs compared to both free ODN and cationic lipid-ODN complexes. Nuclear uptake peaks at 4 h and intact ODN persists in the nucleus with a half-life of 12 h. ODN concentrations of 20-70 microM are achieved at 24 h in all monolayer cell lines evaluated to date. ODNs are detected in 50-100% of the total cell population by immunohistochemistry with apparent uptake into vesicles and nuclear localization. Luciferase expression of a co-delivered reporter plasmid suggests that these ODNs are free in the nucleus. AdpL-ODN complexes will provide a valuable tool for delivering unmodified ODNs to the nucleus of malignant cells.

Influence of GC and AT specific DNA minor groove binding drugs on intermolecular triplex formation in the human c-Ki-ras promoter.

We have used DNase I footprinting and gel shift assays to characterize the interaction of DNA binding drugs mithramycin, distamycin, and berenil with an intermolecular triplex formed by the human c-Ki-ras promoter. A purine-rich triplex-forming oligonucleotide (ODN) forms a stable intermolecular triple helix (triplex) with a homopurine (PR):homopyrimidine (PY) motif in the human c-Ki-ras promoter which contains a 22bp PR:PY region (-328 to -307). This triplex structure is comprised of 15 G.G:C triplets interspersed with 7 T.A:T triplets. Mithramycin binding sites in the human c-Ki-ras promoter encompass most of the triplex target site and three G-C-rich sequences downstream of this triplex-forming region. Mithramycin binding within the c-Ki-ras promoter completely abrogates triplex formation. Furthermore, the addition of mithramycin to pre-formed triplex by c-Ki-ras promoter displaces the major groove bound ODN. Five prominent distamycin binding sites are noted within the c-Ki-ras promoter including the triplex-forming site as well as A-T-rich regions upstream and downstream of the triplex site. Berenil does not bind within the triplex target sequence, and only one berenil binding sequence downstream of the triplex motif was present within the c-Ki-ras promoter fragment. Neither distamycin nor berenil prevents triplex formation, and, furthermore, the addition of either distamycin or berenil to the pre-formed triplex structure did not displace the major-groove-bound third strand. This study demonstrates that GC-specific and AT-specific minor groove ligands differentially affect the intermolecular pur.pur:pyr triplex. A possible biological significance of mithramycin interaction with intramolecular triplex is discussed.

Triplex formation by the human Ha-ras promoter inhibits Sp1 binding and in vitro transcription.

The central role of the ras oncogenes in the pathogenesis of a wide variety of human malignancies is well established. Toward developing specific transcriptional inhibitors of the human Ha-ras oncogene, we have designed oligonucleotides to target a region of the Ha-ras promoter (-8 to -28) which contains two of the three Sp1 binding sites essential for transcriptional activity. Gel mobility analysis and DNase I footprinting demonstrate that an oligonucleotide (HR21ap) forms a sequence-specific triple helix with its target site in an antiparallel orientation with respect to the purine-rich duplex strand through predominantly G*G:C triplets. Within the Ha-ras promoter, HR21ap binds exclusively to the proximal target Sp1 sites over a similar nontarget distal sequence which, like the target, contains a consensus Sp1 site. Protein binding assays demonstrate that triplex formation by HR21ap inhibits Sp1 binding to the Ha-ras promoter. Moreover, oligonucleotide-directed triplex formation arrests Ha-ras promoter-dependent transcription in vitro. The results presented here suggest that triplex formation by the Ha-ras promoter targeted oligonucleotide may provide a means to specifically inhibit transcription of this oncogene in vivo.

Assessing the nicotine content of smokeless tobacco products.

The nicotine content of 11 popular brands of smokeless tobacco--including moist snuff, plug and loose-leaf chewing tobacco--was analyzed. In general, moist snuff has the highest nicotine content and loose-leaf chewing tobacco has the lowest, with plug tobacco falling in the middle. Variability in nicotine content may affect smokeless tobacco use and should be considered when studying usage as a variable for adverse effects of ST use.

Effect of abasic linker substitution on triplex formation, Sp1 binding, and specificity in an oligonucleotide targeted to the human Ha-ras promoter.

A region of the human Ha-ras promoter (-8 to -28) which contains two of the three Sp1 binding sites essential for transcriptional activity forms a sequence specific oligonucleotide-directed pur*pur:pyr triple helix. The relative binding of oligonucleotides containing different substitutions, including an abasic propanediol linker, over three potentially destabilizing C:G interruptions in the otherwise poly G:poly C target was examined. DNase I footprint titrations reveal that substitution of the positively charged abasic propanediol linker results in approximately ten fold greater binding than cytosine substitution which in turn provides greater sequence specific binding than substitution of a guanine in the third strand oligonucleotide over the C:G interruptions. Protein binding assays demonstrate that triplex formation by the linker substituted oligomer (HR21Xap) is less effective in inhibiting Sp1 binding than the cytosine substituted oligomer (HR21ap) both to the target sequence as well as an upstream sequence. As an indication of the effect of linker substitution and targeting consensus Sp1 sites on triplex specificity, the relative ability of the Ha-ras promoter targeted oligonucleotides to interact with non-target Sp1 sequences within the Ha-ras promoter as well as in the DHFR promoter and HIV-1 LTR was also investigated. At concentrations which afford complete DNase I protection of the target sequence, HR21ap does not bind to the non-target sequences while HR21Xap interacts weakly only at a distal site in the DHFR promoter. Also, HR21ap as well as HR21Xap are specific in their inhibition of Sp1 binding. These results suggest that the propanediol linker is able to skip over interruptions in a target sequence thereby stabilizing triplex but, slightly compromises sequence specificity and the ability to inhibit Sp1 binding to the Ha-ras promoter.

Inhibition of nuclear protein binding to the human Ki-ras promoter by triplex-forming oligonucleotides.

The human Ki-ras promoter contains a 22 base pair homopurine.homopyrimidine (pur.pyr) motif within a region that is nuclease-hypersensitive in both native chromatin and supercoiled plasmids. Gel mobility shift analysis and competition experiments show that this pur.pyr motif binds a nuclear protein(s) in a sequence-specific manner. Several observations suggest that the nuclear protein may be an important regulatory factor. Gel mobility shift analysis and DNase I footprinting demonstrate that oligonucleotides can be targeted to this motif forming sequence-specific purine*purine.pyrimidine (pur*pur.pyr) or mixed purine/pyrimidine*purine-pyrimidine (pur/pyr*pur.pyr) intermolecular triple helices through guanine (G) recognition of guanine.cytosine (G.C) base pairs and either adenine (A) or thymine (T) recognition of adenine-thymine (A.T) base pairs in the target sequence. Triple helices containing either T*A.T or A*A.T triplets are formed exclusively with oligonucleotides antiparallel to the homopurine target strand. The affinity of an oligonucleotide which forms T*A.T triplets is approximately equal to, or slightly greater than, the affinity of an oligonucleotide which forms A*A.T triplets. Oligonucleotide-directed triplex formation inhibits sequence-specific nuclear protein binding to the K-ras promoter. These observations suggest that triplex formation by the oligonucleotides described here may provide a means to specifically inhibit transcription of the K-ras oncogene.

The G-C specific DNA binding drug, mithramycin, selectively inhibits transcription of the C-MYC and C-HA-RAS genes in regenerating liver.

Expression of the c-myc and c-Ha-ras protooncogenes is dramatically increased in regenerating rat liver as an early response to partial hepatectomy. Nuclear runon transcription studies confirm that the increased c-myc and c-Ha-ras mRNA levels in regenerating livers reflect transcriptional activation of these genes. Mithramycin, a G-C specific DNA binding drug, prevents the increased transcriptional activity of c-myc and c-Ha-ras genes after hepatectomy but does not alter the transcriptional activity of the beta-actin gene. Continuous exposure of rats to mithramycin after hepatectomy prevents the increase in both c-myc and c-Ha-ras expression and blocks the increased cellular proliferation characteristic of regeneration. The delayed increase in c-myc and c-Ha-ras gene expression is associated with a delay in cellular proliferation. The inhibition of c-myc and c-Ha-ras transcription by mithramycin, the delay in cellular proliferation, and the ability of mithramycin to prevent protein binding to the c-myc promoter, suggest that the increased expression of these genes is a necessary component of liver regeneration.

Triplex formation inhibits HER-2/neu transcription in vitro.

Triplex-forming oligonucleotides (TFOs) have been shown to bind to target DNA sequences in several human gene promoters such as the c-myc oncogene, the epidermal growth factor receptor, and the dihydrofolate reductase genes. TFOs have been shown to inhibit transcription in vitro and gene expression in cell culture of the c-myc and other genes. The HER-2/neu oncogene, which is overexpressed in breast cancer and other human malignancies, contains a purine-rich sequence in its promoter, which is favorable for purine:purine:pyrimidine (R:R:Y) triplex formation. Although its function in the HER-2/neu promoter is unknown, this purine-rich site is homologous to a protein-binding sequence in the promoter of the epidermal growth factor receptor that is necessary for efficient transcription of this gene. We have shown that this sequence is a site for nuclear protein binding by incubation with a crude nuclear extract. We describe the formation of an interstrand triplex using a purine-rich oligonucleotide antiparallel to this purine-rich target sequence of the HER-2/neu promoter. Triplex formation by the oligonucleotide prevents protein binding to the target site in the HER-2/neu promoter in vitro. We have shown that this oligonucleotide is a potent and specific inhibitor of HER-2/neu transcription in an in vitro assay. The triplex target site contains a single pyrimidine base that does not conform to the R:R:Y triplex motif. In an attempt to abrogate the potentially destabilizing effects of this pyrimidine base on triplex formation, we have substituted an abasic linker for the pyrimidine residue in the triplex forming oligonucleotide. Triplex formation with the modified oligonucleotide appears to occur with approximately equivalent binding affinity. Triplex formation in the HER-2/neu oncogene promoter prevents transcription in vitro and may represent a future modality for specific inhibition of this gene in vivo.

Enzyme selectivity analyses of arylsulfonylamino acid aldose reductase inhibitors.

Arylsulfonylamino acids, displaying a wide range of inhibitory activities versus rat lens aldose reductase (RLAR), were analyzed for enzyme selectivity in several test systems. These RLAR inhibitors were found not to produce significant inhibition of genetically-linked reductases (aldehyde reductase, ALR), catalytically similar reductases (Pachysolen tannophilus xylose reductase, PTXR), functionally distinct oxidoreductases (glutathione reductase, GR, lactate dehydrogenase, LDH, and gamma-transaminase, GABA-T), and thymidylate synthase (TS). These data suggest that aldose reductase differs significantly from other oxidoreductases in its inhibitor binding domain(s). Furthermore, the aldose reductase selectivity demonstrated by the arylsulfonylamino acids suggests that these compounds may not inhibit other key metabolic transformations in various cell types and that they may function as selective probes for studies of the relationship between aldose reductase mediated biochemical changes and the pathologies of chronic diabetes.

Relative structure-inhibition analyses of the N-benzoyl and N-(phenylsulfonyl) amino acid aldose reductase inhibitors.

A number of N-benzoyl amino acids were synthesized and tested to compare structure-inhibition relationships with the isosteric N-(phenylsulfonyl) amino acid (PS-amino acid) aldose reductase inhibitors. Inhibition analyses with these series reveals that their kinetic mechanisms of inhibition are similar, but that significant differences in structure-inhibition relationships exist. For example, while the PS-alanines and PS-2-phenylglycines produce enantioselective inhibition (S greater than R), no consistent pattern of enantioselectivity is observed with the isosteric N-benzoylalanines and 2-phenylglycines. Also, N-methyl and N-phenyl substitution in the PS-amino acid series does not substantially alter inhibitory activity, while similar substitutions in the N-benzoyl series (particularly N-phenyl) results in a significant increase in inhibitory activity. Proton NMR analysis of the N-benzoylsarcosines reveals that these compounds exist as a mixture of rotamers in solutions including the enzyme assay buffer and that the preferred conformer is one in which the carboxymethyl moiety is trans to the aromatic ring. Similar analyses with the N-benzoyl-N-phenylglycines demonstrate that these derivatives exist exclusively in the trans rotameric conformation in solution. No such N-substituent effects on conformation were observed in the PS-amino acid series. These results suggest that the differences in structure-inhibition trends between these structurally related series may result from the effect of substituents on preferred conformation.