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In brief: We assessed life events, depression, and general well-being (health concerns, energy, life satisfaction, cheerfulness, tension, and emotional control) in 73 members of the US Alpine Ski Team. We compared these psychological factors with subsequent surveys of general health, illness, and athletic performance and found that psychological status (especially general well-being) was associated with subsequent health problems, injuries, and performance levels. These findings indicate that coaches and sports medicine specialists must be sensitive to psychological disorders in elite athletes and provide appropriate clinical assessment and treatment.

The isolation of pure nucleic acids from clinical samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid testing (NAT). In this study, novel flat glass devices (cards) were demonstrated to support the rapid and efficient extraction of nucleic acids from upper respiratory tract specimens (nasal washes and swabs). The performance of the nucleic acid extraction cards was directly compared to an existing standardized and automated platform for viral extraction from these types of specimens. The flowthrough card method improved the speed of nucleic acid purification and accommodated larger sample volumes in extraction of bacteriophage MS2 RNA from the various specimen matrices. The dynamic range and estimated sensitivity of the card extraction method for reverse transcriptase quantitative real-time PCR (RT-qPCR)-based detection approximate those of the standardized magnetic glass bead extraction method used in this study.

BACKGROUND: Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.

Article 16 of the Law of the Sea Convention substantially expands preexisting international law requirements for the publication of internal water closing lines (collectively referred to herein as “artificial baselines”). Surprisingly, there seems to be substantial confusion among experts as to what exactly is required. For example, most experts appear to conclude that Article 16 requires that artificial baselines be either charted or described by geographic coordinates. This article reviews the language, history, and purposes of Article 16 and concludes that its requirements are more complicated. It then considers which artificial baselines are included within the requirement and what legal consequences evolve from a coastal State's failure to comply with the publicity obligations.

It is well known that DNA strands bind to silica surfaces in the presence of high concentrations of chaotropic salts. We developed simple methods to evaluate binding and recovery of DNA on flat glass microscope slides and compared their properties with commercially available silica "spin columns". Surprisingly, genomic DNA was recovered efficiently from untreated glass slides. Binding and elution times from glass slides were optimized in experiments with DNA samples of various sizes and defined buffers. Average DNA recovery from 500 ng of input genomic DNA varied from 25 to 53% for the glass slide protocol. Yields were comparable to those of spin columns. Human serum albumin and plasma components decreased DNA binding to glass several-fold in a concentration-dependent manner. These results support the concept of using flat glass slides as DNA purification surfaces in microfluidic devices for PCR sample preparation.

BACKGROUND Prostate specific membrane antigen (PSMA) is expressed on the plasma membrane of normal prostate and in primary and metastatic prostate cancer in humans. Recently, a mouse homolog of PSMA (FOLH1) was identified that shares an 85% sequence homology with human PSMA. The transgenic adenocarcinoma of the mouse prostate (TRAMP) model displays spontaneous tumor development with age and metastasizes to tissues similar to human prostate cancer. This study characterized the expression of Folh1 in the TRAMP model to determine if the TRAMP would be a useful model system to evaluate PSMA-directed targeting strategies. METHODS A sensitive, real-time quantitative PCR assay was developed to measure Folh1 cRNA copy number in various tissues of 30–32-week-old TRAMP+ and age-matched, nontransgenic controls (TRAMP−). RESULTS Of the tissues studied, the highest expression of Folh1 was observed in the kidney and brain of both TRAMP+ and TRAMP− mice. Low levels of Folh1 cRNA (1–2 copies/ng total RNA) were detected in the tumor and lymph nodes of TRAMP + mice and in the seminal vesicles and lung of the TRAMP + and TRAMP− mice. The expression of Folh1 mRNA was sixfold higher in the prostate of 32-week-old TRAMP− mice compared to the tumor of 32-week-old TRAMP + mice. The rank order of the Folh1 expression in the tissues studied was kidney > brain > prostate > tumor > lymph nodes > lung > seminal vesicles > liver. Folh1 mRNA was undetectable in the bone marrow of both TRAMP+ and TRAMP− mice. Folate hydrolase activity assayed in the kidney, brain, lung, and liver paralleled the expression of Folh1 mRNA in these tissues. CONCLUSIONS We demonstrate that Folh1 is expressed at very high levels in some normal mouse tissues including the prostate gland and that the expression is not upregulated in the tumor of 32-week-old TRAMP + mice. Prostate 55: 308–316, 2003. © 2003 Wiley-Liss, Inc.

Triplex forming oligonucleotides (TFOs) have the ability to site specifically modulate gene expression through the formation of triple helix DNA. The HER-2/neu promoter contains a strategically located triplex target sequence, and has been successfully targeted in vitro, with little success in vivo. A TFO was conjugated at both its 5' and 3' ends to an alkylating agent (phenylacetate mustard) in an attempt to stabilize the triple helix intracellularly. In vitro assays demonstrated that the bis-conjugate bound the duplex and alkylated the target guanine residues with high efficiency. The bis-conjugate suppressed promoter activity by 60-70% in cancer cells using a plasmid with a preformed triple helix, and the suppression was minimal when the nitrogen mustard was conjugated at only one end. Helicase assays demonstrated that helicase activity can unwind the TFO at the unalkylated end of the triple helix, which may leave the unwound oligonucleotide susceptible to nuclease degradation or ineffective at inhibiting transcription initiation. Our findings indicate that dual alkylation of the target sequence is required to suppress the intracellular activity of a reporter plasmid with a preformed triple helix, likely due to greater stability of the triple helix within cells and inhibition of helicase activity.

Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson-Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs. In the PPG heterocycle, the N-7 and C-8 atoms of G are interposed. This leaves the ring system with an electron density similar to G, but prevents Hoogsteen-bonding associated with N-7. ODNs containing multiple PPG bases were easily prepared using a dimethylformamidine-protected phosphoramidite reagent. Substitution of PPG for G in ODNs allowed formation of more stable DNA duplexes. When one or more PPGs were substituted for G in ODNs containing four or more consecutive Gs, G aggregation was eliminated. Substitution of PPG for G also improved discrimination of G/A, G/G and G/T mismatches in Watson-Crick hybrids. Use of PPG in fluorogenic minor groove binder probes was also explored. PPG prevented aggregation in MGB probes (MGB(TM) is a trademark of Epoch Biosciences) and allowed use of G-rich sequences. An increased signal was observed in 5'-PPG probes due to reduced quenching of fluorescein by PPG. In summary, substitution of PPG for G enhances affinity, specificity, sensitivity and predictability of G-rich DNA probes.

Attachment of oligodeoxynucleotides (ODNs) containing benzaldehyde (BAL) groups to semicarbazide-coated glass (SC-glass) slides is described. 5'-BAL-ODNs are prepared using automated DNA synthesis and an acetal-protected BAL phosphoramidite reagent. The hydrophobic protecting group simplifies purification of BAL-ODNs by reverse phase HPLC and is easily removed using standard acid treatment. The electrophilic BAL-ODNs are stable in solution, but react specifically with semicarbazide groups to give semicarbazone bonds. Glass slides were treated with a semicarbazide silane to give SC-glass. BAL-ODNs are coupled to the SC-glass surface by a simple one-step procedure that allows rapid, efficient and stable attachment. Hand-spotted arrays of BAL-ODNs were prepared to evaluate loading density and hybridization properties of immobilized probes. Hybridization to radiolabeled target strands shows that at least 30% of the coupled ODNs were available for hybridization at maximum immobilization density. The array was used to probe single nucleotide polymorphisms in synthetic DNA targets, and PCR products were correctly genotyped using the same macroarray. Application of this chemistry to manufacturing of DNA microarrays for sequence analysis is discussed.

Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.

Antigene radiotherapy, a procedure based on delivery of short-range Auger-electron-emitting radioisotopes to target genes via sequence-specific triplex-forming oligonucleotides, has been successfully demonstrated in vitro using the well-studied radionuclide 125I. To proceed with in vivo trials, Auger electron emitters with shorter half-lives than 125I are required. Here we report a study of the efficiency and distribution of sequence-specific DNA strand breaks produced by decay of 123I and mIIn. 123I and 111In were introduced into triplex-and duplex-forming oligodeoxyribonucleotides (ODNs) through carbohydrate linkers of various lengths. Labeling with radioiodine was performed through tributylstannylbenzamide intermediates while 111In was attached via DTPA. The Auger-emitter-labeled ODNs were hybridized to a single-stranded DNA target, to form duplexes. After decay accumulation, the target DNA samples were assayed for strand breaks using a sequencing gel-electrophoresis technique. For the first time, we observed footprints of DNA strand breaks produced by 123I and 111In. Most of the breaks were located within 10 nucleotides from the decay site. The yield of strand breaks per decay varies; decay of 111In breaks DNA almost 10 times more effectively than decay of 123I. Both 123I and 111In are less effective in breaking DNA strands than 121I, which reflects the higher total energy of the Auger decay process of 125I.

A method utilizing electroporation to deliver antisense oligodeoxynucleotides into Paramecium tetraurelin has been developed. For these studies antisense oligonucleotides directed to different regions of the calrnodulin mRNA were used. It was found that a pulse delivered at 150-250 V ( 375-625 V/cm field strength) for 3.9-4.2 ms using a 275 pF capacitor with resistance set at 13 Ohms was sufficient to achieve measurable incorporation of fluorescently-labeled oligodeoxynucleotides in up to 95% of the cells treated. Optimal parameters included using oligodeoxynucleotides of at least 12 bases in length with a 3' blocking group at a dose of around 10 )LM. In addition, multiple oligodeoxynucleotides directed to the same target rnRNA resulted in at least a 10-fold reduction in the dose of oligodeoxynucleotide required to achieve the desired effects. Taken together, these results indicate that the use of antisense oligodeoxynucleotides can be an easy and useful method for linking genes to specific functions in Paramecium tetraurelia. Finally, this report discusses how different 3' blocking groups and the use of combinations of oligodeoxynucleotides directed to different regions of the same target mRNA can help address concerns about specificity. Supplementary key words. Calcium-dependent K' channel, calcium-dependent Na' channel, calmodulin, cholesterol-linked oligo- deoxynucleotides, hexanol-linked oligodeoxynucleotides, protozoan behavior. NTISENSE oligodeoxynucleotides (ODN) can be used to A transiently down-regulate a gene's expression and thereby help identify the role of the gene product in the cell (2). Their ease of use makes antisense ODN an attractive method for uti- lizing a "reverse genetics" approach for the assignment of function to cloned genes. Previously, we described the micro- injection of antisense ODN as a viable methodology in the cil- iated protozoan, Paramecium tetraurelia (5). In that report, an- tisense ODN were targeted against calmodulin mRNA resulting in abbreviated behavioral responses that mimicked the type of behavioral responses seen in cells with a mutant calmodulin gene that does not properly activate the calcium-dependent Na+ current. Thus, it was concluded that the antisense ODN reduced the levels of calmodulin sufficient to affect the activation of the calcium-dependent Na+ current. Recently it was reported that electroporation can be used as a method for incorporating ODN into cells (l) and, in an effort to improve the ease of use of this technology in ciliates, we have developed a procedure for introducing antisense ODN by electroporation. Calmodulin was again chosen as the target be- cause of its well-characterized role in the control of behavior in Paramecium (6, 141. In addition, we have characterized some of the parameters important for the use of this methodology in Paramecium.

Triplex-forming oligonucleotides (TFOs) bind in the major groove to specific double-helical DNA sequences and have been shown to inhibit the function of targeted genes. Diaziridinylquinones are bifunctional alkylating agents that can form interstrand cross-links in DNA at 5‘-GNC sites. To demonstrate the feasibility of targeted interstrand cross-linking, a diaziridinylquinone−TFO conjugate was prepared from a diaziridinylquinone intermediate bearing an activated ester linker. The first demonstration of triplex-directed interstrand DNA cross-linking by a single targeted bifunctional alkylating agent is described. Up to 38% interstrand cross-linking was observed at pH 6.2.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.

A versatile method is described for preparing aryl nitrogen mustard-oligodeoxyribonucleotide (mustard-ODN) conjugates under anhydrous conditions. The chemistry uses DMSO soluble triethylammonium or tributylammonium salts of the ODNs. A G/A motif triplex forming ODN was chosen for study since it had been shown earlier to bind with high affinity and specificity to a duplex DNA target. A 5'-hexylamine derivative of this ODN was reacted with three different 2,3,5,6-tetrafluorophenyl ester derivatives of aryl nitrogen mustards which were designed to have different alkylation rates. An HPLC assay was used to determine reaction rates of these mustard-ODNs under various conditions. The reactivity of the mustard groups depended on chloride concentration and the presence of nucleophiles. Conjugation of mustards to G/A-containing ODNs decreased their aqueous stability. Hydrolysis and alkylation rates of these agents were consistent with reaction via an aziridinium intermediate. Rates of sequence specific alkylation within a triplex were determined by denaturing gel electrophoresis and shown to depend on inherent reactivity of the mustard group. The improved synthesis and chemical characterization of mustard-ODNs should facilitate their use as sequence specific alkylating agents and as probes for nucleic acid structure.

We studied the yield and distribution of DNA strand breaks produced by decay of 125I introduced into triplex-forming and duplex-forming oligodeoxyribonucleotide (ODNs) through linkers of various lengths. ODNs were prepared with 125I attached at the 5'-end with a long linker or to an internal nucleotide position with a short linker. The 125I-ODNs were hybridized to either a single-stranded target to form duplexes or to a double-stranded target to form triplexes. After decay accumulation, the duplex and triplex samples were assayed for strand breaks in a sequencing gel. The yield of strand breaks per decay was 0.34 for duplex with the 5'-modified ODN and 0.66 for duplex with internally modified ODN. The triplex samples with internal 125I have different yields of DNA breaks in the pyrimidine and purine strands, 0.16 and 0.37, respectively. The yield of DNA breaks in the pyrimidine strand of the triplex with the 5'-modified ODN is 0.46. The majority of breaks are located within 5 nucleotides from the decay site. The yield of strand cleavage per decay of 125I was nearly two-fold lower with the described linkers in comparison with the results obtained when 125I is directly attached to the C-5 position of cytosine. Nevertheless, the rapid iodination procedure reported here combined with the possibility of multiple incorporations of 125I on the linkers makes such 125I-ODNs promising agents for sequence-specific cleavage of DNA.

Apparent equilibrium association constants were determined by gel mobility shift analysis for triple strand formation between a duplex target containing a 21 base long A-rich homopurine run and several end-modified C(m)/T (pyrimidine motif; C(m) = 5-methylcytosine), G/A (purine motif), and G/T (purine-pyrimidine motif) triplex-forming oligonucleotides (TFOs). Incubations were carried out for 24 h at 37 degrees C in 20 mM HEPES, pH 7.2, 10 mM MgCl2, and 1 mM spermine. The purine motif triplex was the most stable (Ka = 6.2 x 10(8) M-1) even though the TFO self-associated as a linear duplex. Conjugation of a terminal hexanol or cholesterol group to the G/A-containing TFO reduced triplex stability by 1.6- or 13-fold, whereas an aminohexyl group or intercalating agent (acridine or psoralen) increased triplex stability by 1.3- or 13-fold. These end groups produced similar effects in C(m)/T and G/T triplexes, although the magnitude of the effect sometimes differed. Addition of 140 mM KCl to mimic physiological conditions decreased stability of the G/A triplex by 1900-fold, making it less stable than the C(m)/T triplex. The inhibitory effect of KCl on G/A triplex formation could be partially compensated for by conjugating the TFO to an intercalating agent (30-350-fold stabilization) or by adding the triplex selective intercalator coralyne (1000-fold stabilization). Although the G/T triplex responded similarly to these agents, the stability of the C(m)/T triplex was unaffected by the presence of coralyne and was only enhanced 1.4-2.8-fold when the TFO was linked to an intercalating agent. In physiological buffer supplemented with 40 microM coralyne, the G/A triplex (Ka = 3.0 x 10(8) M-1) was more stable than the C(m)/T and G/T triplexes by factors of 300 and 12, respectively.

Oligodeoxyribonucleotides (ODNs) with conjugated reactive groups are potential sequence-specific gene inactivating agents. The antitumor antibiotic CC-1065 binds preferably in the minor groove of A-T-rich sites of double-stranded DNA, and the cyclopropapyrroloindole (CPI) subunit of the drug alkylates adenines at their N3 position. Pure enantiomeric (+)- and (−)-CPI and its N5-methyl homologue (MCPI) were synthesized and conjugated to an ODN. These conjugates were evaluated for their ability to alkylate a target containing a duplex region immediately adjacent to a single-stranded complementary binding region for the ODN conjugate. The conjugates demonstrated excellent stability in physiologic conditions and stereospecific, hybridization-triggered alkylation of the synthetic ODN targets. The dependence of the reaction rates on sequence of the duplex target region was in accord with the predicted minor groove binding of the conjugated CPI. The reactivity was highly dependent on the structure of the cross-li...

A rapid and efficient method for the synthesis of 125I-labeled oligodeoxynucleotides ([125I]ODNs) is described. The key intermediates are tributylstannylbenzamide-modified ODNs (Sn-ODNs). Reaction conditions are described for the preparation of 5'-modified Sn-ODNs. Treatment with NaI and chloramine T gave conversion to the desired I-ODN, which was easily isolated by reversed phase chromatography. Thermal denaturation (Tm) studies showed that hybridization properties were not disturbed by the 4-iodobenzamide modification. An [125I]ODN was prepared and characterized by hybridization to 32P-labeled DNA targets. Sequence specific cleavage of the target DNA strand by 125I was measured.

Short DNA duplexes with cholesterol linked at the 3'-terminus of each strand have unique, selective cytotoxic properties. The structural requirements for biological activity were explored through chemical synthesis of analogs and testing in cultured hepatoma cells. Effects of modifications to the sequence, backbone, 3'-sterol, 3'-linker, and 5'-terminus were evaluated. Self-complementary 3'-modified oligodeoxynucleotide (ODN) 10-mers were prepared from solid supports bearing the modification and linker of interest. Any changes to the normal phosphodiester backbone were poorly tolerated. The presence of cholesterol or a closely related sterol was an absolute requirement for activity. The length and position of attachment of the linker to cholesterol was important, with longer linkers showing reduced activity. Large, lipophilic groups at the 5'-terminus gave reduced cytotoxicity and poor solubility properties. The short length and unique structure of these ODNs allowed efficient automated synthesis on a 400 mumol scale and simplified purification.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

Under Article 7 (1) of UNCLOS, the method of straight baselines joining appropriate points may be employed in localities where the coastline is deeply indented and cut into, or if there is a fringe of islands along the coast in its immediate vicinity. In several instances, bays shared by two or more States have not been closed, even though they would meet the geographical conditions required for the closing of uridical bays. The baseline of the Rio de la Plata has given rise to protests by France, the Netherlands, the United Kingdom and the United States. In international practice there are cases where straight closing lines have been drawn by the bordering States concerned; they have not always led to protests by other States; and one of them has been endorsed also by the International Court of Justice. Keywords:France; International Court of Justice; Rio de la Plata; UNCLOS

Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.The objective was to measure pH profiles of bacterially contaminated PCs over 7 days of storage. Small-volume PC storage bags with incorporated pH sensor were prepared and in vitro variables were tested using aliquots of PCs. The pH sensors were used to delineate trends associated with the deterioration of these PCs upon inoculation with 19 different bacterial strains and one yeast.Monitoring the pH trends in real time in a noninvasive fashion, most bacterial strains were detected within 24 to 72 hours after spiking into the bag. At the time of detection, bacterial concentrations had reached levels between 1×10(3) and 1×10(8) colony-forming units/mL. Several strains had pH rebound after initial drop. Multiple noninvasive pH reads allowed bacterial detection whereas single pH reads could give false-negative results.The noninvasive pH sensor facilitated the detection of most strains of bacterial contaminants within 3 days with no potential for sampling error.

The calcium-binding protein calmodulin has been shown to modulate the Ca(2+)-dependent ion channels of Paramecium tetraurelia. Mutations in the calmodulin gene of Paramecium result in an altered pattern of behavioral responses. Antisense oligodeoxyribonucleotides (ODNs), complementary to calmodulin mRNA in Paramecium, were synthesized from a modified solid support that introduced a 3'-hydroxyhexyl phosphate. These 3'-modified ODNs were tested for their ability to alter the behavioral response of Paramecium. The microinjection of antisense ODNs temporarily reduced the backward swimming behavior of the cells in test solutions containing Na+. The injection of sense and random 3'-modified ODNs, or unmodified antisense ODNs, had no effect. The antisense ODN-induced effect was reversed by the injection of calmodulin protein. The pattern of response of the injected cells in various behavioral test solutions indicated that the calmodulin antisense ODNs reduce the Ca(2+)-dependent Na+ current. Antisense ODNs, complementary either to the 5' start site or to an internal sequence of the calmodulin mRNA, were similarly effective in altering behavior. These results show that antisense ODNs may be utilized in ciliated protozoa as a tool for reducing the expression of specific gene products. In addition, Paramecium represents a powerful model system with which to study and develop antisense ODN technology.

BACKGROUND: Stored platelets (PLTs) are metabolically active, resulting in a decrease of pH during storage. The pH of PLT concentrates (PCs) is recognized as a measure of quality, and pH limits are set by regulatory bodies. A pH sensor was built into a PLT storage container, and the feasibility of testing pH using a noninvasive fluorescent measurement method was evaluated. STUDY DESIGN AND METHODS: A citrated polyvinylchloride (PVC) PLT storage container with pH sensor insert was made and evaluated for biocompatibility during PLT storage and on pH reading accuracy, reproducibility, and durability. A noninvasive fluorescence reader was tested versus syringe-based sampling and subsequent measurement with a blood gas analyzer (BGA). The effect of interfering substances in plasma on the accuracy of this optical measurement was tested. Calibration and accuracy of the pH sensor were determined in both phosphate-buffered saline and in PCs. RESULTS: The citrated PVC storage container with pH sensor insert showed good storage properties for 300 mL of pooled buffy coat PLTs in plasma over 7 days. The pH sensor was easy to use and tracked pH22 in the range of 6.2 to 7.8 over 11 days of storage. Accuracy in PCs was 0.08 pH units measured at 22°C when calibrated against a BGA. CONCLUSION: The storage container with integrated pH sensor and noninvasive reader allows pH of PCs to be tracked over time in a noninvasive manner.

Protein sulfhydryl reactive N-(4-[125I]iodophenethyl)maleimide (IPEM, 5) was obtained from N-[4-(tri-n-butylstannyl)phenethyl]maleimide in 59-100% radiochemical yield. Conjugation of 5 to NR-ML-05 Fab, a murine anti-melanoma antibody Fab fragment that had been previously reduced with dithiothreitol (DTT), was effected in an average of 85% yield. Results from in vitro chemical challenges and serum stability studies on the IPEM conjugate of NR-ML-05 Fab (6) indicated a stable covalent attachment of the radioiodine. A biodistribution study of the IPEM conjugate in tumor-bearing athymic nude mice showed lack of significant accumulation of radioiodine in the thyroid and stomach which was an indication of in vivo stability. The observed uptake in tumor was consistent with that obtained for Chloramine-T- or p-iodobenzoate-labeled NR-ML-05 Fab conjugates.

A procedure for immobilization of well-defined quantities of oligodeoxyribonucleotides (ODNs) to a versatile nylon support is described. The solid support, a nylon-6/6 bead, is covalently coated with poly(ethyleneimine) to provide a reactive spacer-arm for attachment of ODNs. 5'-Aminohexyl-tailed ODNs are selectively activated using 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride) and then covalently attached to the bead via the triazine moiety. The modified nylon support has a low level of binding of nonspecific nucleic acid and efficiently captures both RNA and DNA targets.

Various types of minor groove binders have been attached to synthetic oligodeoxynucleotides, and the interactions of these conjugates (MB-ODNs) with DNA are reviewed here. MB-ODNs have enhanced DNA affinity and have improved the hybridization properties of sequence-specific DNA probes. Short MB-ODNs hybridize with ssDNA to give more stable DNA duplexes than unmodified ODNs with similar lengths. Mismatch discrimination of short MB-ODNs is enhanced in comparison to longer unmodified ODNs. The stronger binding of MB-ODNs allows for more stringent hybridization conditions to be used in DNA probe-based assays. MB-ODNs are especially useful in quantitative "real-time" PCR assays since they bind efficiently during the high-temperature primer extension cycle. The synthesis and biophysical chemistry of MB-ODN conjugates are reviewed here. Four published structural classes of MB-ODNs and their various dsDNA binding modes are discussed, and the well-characterized DPI3-type MB-ODNs and their interactions with ssDNA target strands are described in detail.

This unit describes, in detail, the preparation of 3-aminopropyl-substituted pyrazolo[3,4-d]pyrimidine analogs of the purines deoxyadenosine (dA) and deoxyguanosine (dG). Phosphoramidite reagents of these so-called aminopropyl-PPA and -PPG nucleosides (AP-PPA and AP-PPG, respectively) allow introduction of amino linkers into internal positions of synthetic DNA strands. Synthesis of suitably protected AP-PPA and AP-PPG phosphoramidites are described. The stepwise alkynylation, hydrogenation, selective protection, and phosphoramidite synthesis is similar for both the PPA and PPG analogs. To demonstrate the application of these reagents, a protocol is given in which a simple DNA strand is synthesized and conjugated to a lipophilic activated ester (dabcyl-SE) to form a stable amide linkage. Utility of this chemistry for preparing internally modified DNA conjugates is discussed.

This paper describes a study of traffic loads applied to a reinforced concrete bridge during and after its strengthening with externally bonded fiber-reinforced polymer (FRP) reinforcement. In a laboratory, eight beams were tested. Seven of them were strengthened with a precured, unidirectional, carbon-fiber laminate strip that represented the materials used on the actual bridge. The variables tested included the intensity and frequency of load cycles applied during the epoxy-curing period, the thickness of the epoxy layer, and the thickness of the FRP strip. All these beams failed initially with the FRP debonding in the maximum moment region. For all of the traffic loads applied during and after installation no reduction in strengthening was seen. The experiment concluded that concrete bridges can be strengthened effectively even if they remain open to traffic during the procedure.

In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.

4-Alkynyl-3-methoxy-4-hydroxycyclobutenones, a readily available class of compounds, cleave supercoiled DNA by a mechanism that appears to require at least some involvement of diradical intermediates formed in the ring expansion of the cyclobutenones

Fluorogenic 2'-deoxynucleotide probes containing a minor groove binding-quencher compound at the 5'-end and a fluorophore at the 3'-end, were recently described. These probes fluoresce upon hybridization to the complementary target. The 5'-MGB-quencher group prevents 5'-nuclease digestion by Taq polymerase during homogeneous amplification. The 5'-MGB-quencher-oligonucleotide-fluor (MGB-Q-ODN-Fl) probes displayed a dynamic range of 7 order of magnitude, with an ultimate sensitivity of better than 5 copies per sample. The high sensitivity and specificity is illustrated by the application of the probes in single nucleotide polymorphism detection, final load determination and gene expression analyses. This paper summarizes new developments in sequence detection, gene expression and SNP analysis using new Tm prediction software to design robust 5'-MGB-Q-ODN-Fl probes. Furthermore, the software is capable of estimating the Tm of probes containing a modified base. Due to G:G self-association, many G-rich probes and primers are poor performers in amplification reactions. The software recognizes such sequences and substitution of G with 6-Amino-1,5-dihydro-pyrazolo(3,4- d)pyrimidin-4-one (PPG) is indicated, when necessary to eliminate G:G self-association. Examples of improved performance of PPG containing primers and probes is demonstrated.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Triplex-forming oligonucleotides (TFOs) are being investigated as highly specific DNA binding agents to inhibit the expression of clinically relevant genes. So far, they have been shown to inhibit transcription from the HER-2/neu gene in vitro, whereas their use in vivo has been studied to a limited extent. This study uses a TFO-chlorambucil (chl) conjugate capable of forming site-specific covalent guanine adducts within the HER-2/neu promoter. We demonstrate that nucleotide excision repair (NER) represents a mechanism of cellular resistance to TFO-directed DNA alkylation. In vitro repair assays demonstrate that triplex-directed chl-guanine adducts are substrates for repair by NER competent cell extracts but not XP12BE cell extracts deficient in NER. The degree of repair is estimated by a ligation-mediated polymerase chain reaction with a pre-formed triplex in a plasmid transfected into repair competent cells, indicating that approximately 25% of the guanine adducts are removed after 24 h. These data indicate that guanine adducts from TFO-directed alkylation are a substrate for NER and that DNA repair is a significant barrier to the intracellular persistence of target gene binding by TFOs.

Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P:0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.

Publisher Summary This chapter describes two general methods for attaching conjugate groups to oligonucleotides containing alkylamine linker arms. It uses a simple acylation reaction with a variety of activated esters for the preparation of hundreds of oligonucleotide conjugates. The first method (Method A) describes reaction of high-performance liquid chromatography (HPLC)-purified amine-modified oligonucleotides under anhydrous conditions. Triethylammonium (TEA) salts of oligonucleotides are soluble in dimethyl sulfoxide (DMSO) and allow the successful conjugation of lipophilic and hydrolytically unstable groups in homogeneous organic solution. The anhydrous conditions also protect the electrophile from hydrolysis and require only a small excess of conjugate group. This versatile method has been used to prepare all three major types of oligonucleotide conjugates. The second method (Method B) describes acylation of 5'–alkylamine-modifled ofigonucleotides while still immobilized on the solid support. This simpler method has advantages for conjugate groups that can survive oligonucleotide deprotection conditions. The two general methods are compared by preparing a specific oligonucleotide conjugate: an oligodeoxynucleotide (ODN) with a 5'-minor groove-binding (MGB) conjugate group.

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.

We describe sequence-specific alkylation in the minor groove of double-stranded DNA by a hybridization-triggered reactive group conjugated to a triplex forming oligodeoxyribonucleotide (TFO) that binds in the major groove. The 24 nt TFOs (G/A motif) were designed to form triplexes with a homopurine tract within a 65 bp target duplex. They were conjugated to an N 5-methyl-cyclopropapyrroloindole (MCPI) residue, a structural analog of cyclopropapyrroloindole (CPI), the reactive subunit of the potent antibiotic CC-1065. These moieties react in the DNA minor groove, alkylating adenines at their N3 position. In order to optimize alkylation efficiency, linkers between the TFO and the MCPI were varied both in length and composition. Quantitative alkylation of target DNA was achieved when the dihydropyrroloindole (DPI) subunit of CC-1065 was incorporated between an octa(propylene phosphate) linker and MCPI. The required long linker traversed one strand of the target duplex from the major groove-bound TFO to deliver the reactive group to the minor groove. Alkylation was directed by relative positioning of the TFOs. Sites in the minor groove within 4-8 nt from the end of the TFO bearing the reactive group were selectively alkylated.

G/A motif triplex-forming oligonucleotides (TFOs) complementary to a 21 base pair homopurine/homopyrimidine run were conjugated at one or both ends to chlorambucil. These TFOs were incubated with several synthetic duplexes containing the targeted homopurine run flanked by different sequences. The extent of mono and interstrand cross-linking was compared with the level of binding at equilibrium. Covalent modification took place within a triple-stranded complex and usually occurred at guanine residues in the flanking double-stranded DNA. The efficiency of alkylation was dependent upon the sequence of the flanking duplex, the solution conditions, and the rate of triplex formation relative to the rate of chlorambucil reaction. Self-association of the TFOs as parallel duplexes was demonstrated and this did not interfere with triple strand formation. With an optimal target, cross-linking of the triplex was very efficient when incubation was carried in a physiological buffer supplemented with the triplex selective intercalator coralyne.

We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.

An efficient chemical procedure for the immobilization of carboxylate containing conjugate groups onto controlled pore glass (CPG) is described. The derivatized supports were used in the automated synthesis of an oligodeoxynucleotide (20-mer ODN) containing a 3' phosphodiester linked hexanol, aminohexyl, acridine, or cholesterol group. The stability of the oligomer in a hepatoma cell culture was found to be prolonged two to three fold by the presence of any of the 3' tails. By contrast, an aminohexyl group appended to the 5' terminus of the ODN only marginally improved its nuclease resistance. These data support the notion that antisense ODNs are primarily degraded by 3' exonucleases. Introduction of simple 3' tails which incorporate a normal phosphodiester linkage can increase ODN stability by interfering with these enzymes.

A new controlled-pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotides bearing a 3'-aminohexyl tail. This solid support (AH-CPG) exhibits superior performance as compared to a commercially available 3'-amine CPG. The AH-CPG is prepared from 6-aminohexan-1-ol with a unique protecting group for the amine that also functions as the site of attachment to the CPG. A 3'-amine-tailed oligodeoxynucleotide (ODN) was prepared from this support using standard phosphoramidite coupling and deprotection conditions. The 3'-amine-tailed ODN was subsequently modified with an acridinylpropionic acid tetrafluorophenyl ester. Facile synthesis of the AH-CPG and the stability of the deprotected product makes this functionalized solid support especially useful for preparation of oligonucleotides bearing 3'-amine tails and other modifications.

New solid supports are described which allow the direct synthesis of oligonucleotides bearing either cholesterol or acridine at the 3'-terminus. A stereochemically defined amino diol was prepared by reduction of N-Cbz-hydroxy-L-proline. This linker molecule was first acylated with the desired conjugate molecule, then protected as the dimethoxytrityl ether. The remaining secondary hydroxyl group was succinylated and immobilized on a controlled-pore glass support. 3'-Modified oligodeoxynucleotides (ODNs) were prepared from these supports by using standard phosphoramidite coupling and deprotection conditions. A cholesterol-modified support was prepared from cholesterol chloroformate and the amino diol linker. Two types of acridine-modified solid supports were prepared from acridine tetrafluorophenyl esters with linker arms of different length. In an alternative synthesis of 3'-derivatized ODNs, these active esters were also utilized for acylation of a 3'-amine-modified ODN. A thermal denaturation study was done to determine the effect of the different linker arms on hybridization to a complementary ODN target. Facile synthesis and purification of the 3'-modified ODNs makes these functionalized solid supports especially useful for preparation of oligonucleotides bearing these and other modifications.

During 19771978 a series of 25 ft wide 8 m by 30 ft deep 9 m sewer culverts were built in the Islais Creek Basin near San Francisco Bay. The sheetpile wall supported excavations for the culverts we...