Trophoblast expression of the minor histocompatibility antigen HA-1 is regulated by oxygen and is increased in placentas from preeclamptic women.

INTRODUCTION: Maternal T-cells reactive towards paternally inherited fetal minor histocompatibility antigens are expanded during pregnancy. Placental trophoblast cells express at least four fetal antigens, including human minor histocompatibility antigen 1 (HA-1). We investigated oxygen as a potential regulator of HA-1 and whether HA-1 expression is altered in preeclamptic placentas. METHODS: Expression and regulation of HA-1 mRNA and protein were examined by qRT-PCR and immunohistochemistry, using first, second, and third trimester placentas, first trimester placental explant cultures, and term purified cytotrophoblast cells. Low oxygen conditions were achieved by varying ambient oxygen, and were mimicked using cobalt chloride. HA-1 mRNA and protein expression levels were evaluated in preeclamptic and control placentas. RESULTS: HA-1 protein expression was higher in the syncytiotrophoblast of first trimester as compared to second trimester and term placentas (P<0.01). HA-1 mRNA was increased in cobalt chloride-treated placental explants and purified cytotrophoblast cells (P = 0.04 and P<0.01, respectively) and in purified cytotrophoblast cells cultured under 2% as compared to 8% and 21% oxygen (P<0.01). HA-1 mRNA expression in preeclamptic vs. control placentas was increased 3.3-fold (P = 0.015). HA-1 protein expression was increased in syncytial nuclear aggregates and the syncytiotrophoblast of preeclamptic vs. control placentas (P = 0.02 and 0.03, respectively). DISCUSSION: Placental HA-1 expression is regulated by oxygen and is increased in the syncytial nuclear aggregates and syncytiotrophoblast of preeclamptic as compared to control placentas. Increased HA-1 expression, combined with increased preeclamptic syncytiotrophoblast deportation, provides a novel potential mechanism for exposure of the maternal immune system to increased fetal antigenic load during preeclampsia.

Short communication: relationships between α-lactalbumin and quality traits in bulk milk.

The main objective of this study was to investigate whether the α-lactalbumin (α-LA) content of bulk milk is related with some known inflammatory markers and milk quality traits. An additional objective was to study whether combining α-LA, haptoglobin (Hp), and serum amyloid A (SAA) in an acute phase index (API) could be useful as an alternative marker for bulk milk quality. For the dairy industry, it is of great importance to receive high quality bulk milk for manufacture of liquid milk and dairy products. The somatic cell count (SCC) is currently used as an indirect marker for bulk milk quality, but because it is somewhat insensitive and unspecific, interest exists in alternative markers. Bulk milk samples were analyzed for α-LA, SCC, polymorphonuclear leukocyte count, Hp, SAA, fat, lactose, total protein and casein contents, casein number, protein composition, proteolysis, and coagulating properties. No significant differences were found in SCC, polymorphonuclear leukocyte count, Hp, or SAA between milk samples containing low, medium, or high concentrations of α-LA. Differences between α-LA groups were, however, found in some milk quality traits because high α-LA concentration was related to low concentrations of α(S1)-, α(S2)-, and ß-caseins and high concentrations of lactose and ß-lactoglobulin. A high API was related to low lactose content and casein number. Samples with high SCC contained less casein and had a lower casein number than milk with a low SCC, and proteolysis was significantly higher in high SCC milk than in low SCC milk. Neither α-LA nor API proved to be a better marker than SCC for the quality traits investigated, and α-LA was not considered to be a useful inflammatory marker in bulk milk.

Radiohybridization PET imaging of KRAS G12D mRNA expression in human pancreas cancer xenografts with [(64)Cu]DO3A-peptide nucleic acid-peptide nanoparticles.

There is a compelling need to image pancreas cancer at an early stage. Human pancreas cancer cells display elevated levels of KRAS protein due to high copy numbers of KRAS mRNA, and elevated levels of insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. Therefore we hypothesized that pancreas cancer could be detected in vivo with a single probe that targets both KRAS mRNA and IGF1R. Because positron emission tomography (PET) is a sensitive imaging technique, we designed a probe incorporating the positron-emitting nuclide (64)Cu. The KRAS-specific hybridization probe consisted of 1,4,7-tris(carboxymethylaza)cyclododecane-10-aza-acetyl (DO3A) on the N-terminus of a peptide nucleic acid (PNA) hybridization sequence (GCCATCAGCTCC) linked to a cyclized IGF1 peptide analog (d-Cys-Ser-Lys-Cys) on the C-terminus, for IGF1R-mediated endocytosis. A series of such KRAS radiohybridization probes with 0, 1, 2 or 3 mismatches to KRAS G12D mRNA, including exact matches to wild type KRAS mRNA and KRAS G12V mRNA, along with a double d(Ala) replacement IGF1 peptide control, were assembled by continuous solid phase synthesis. To test the hypothesis that KRAS-IGF1 dual probes could specifically image KRAS mRNA expression noninvasively in human IGF1R-overexpressing AsPC1 pancreas cancer xenografts in immunocompromised mice, [(64)Cu]PNA radiohybridization probes and controls were administered by tail vein. The [(64)Cu]KRAS-IGF1 radiohybridization probe yielded strong tumor contrast in PET images, 8.6 +/- 1.4-fold more intense in the center of human pancreas cancer xenografts than in the contralateral muscle at 4 h post-injection. Control experiments with single base KRASmismatches, an IGF1 peptide mismatch, and a breast cancer xenograft lacking KRAS activation yielded weak tumor contrast images. These experiments are consistent with our hypothesis for noninvasive PET imaging of KRAS oncogene expression in pancreas cancer xenografts. Imaging oncogene mRNAs with radiolabel-PNA-peptide nanoparticles might provide specific genetic characterization of preinvasive and invasive pancreas cancers for staging and choice of therapy.

Receptor-mediated internalization of chelator-PNA-peptide hybridization probes for radioimaging or magnetic resonance imaging of oncogene mRNAs in tumours.

Early external detection of cancer gene activity might enable early treatment of cancer and might reduce cancer mortality. We hypothesized that oncogene mRNA overexpressed at thousands of copies per malignant cell in a zone of transformed cells could be imaged externally by scintigraphic imaging, PET (positron emission tomography) or MRI (magnetic resonance imaging) with PNA (peptide nucleic acid) hybridization probes that include chelators for metal cations and a cyclized peptide analogue of IGF-1 (insulin-like growth factor 1), D(Cys-Ser-Lys-Cys), to mediate internalization by IGF1R (IGF-1 receptor) overexpressed on cancer cells. We observed that human MCF7 breast cancer cells that overexpress IGF1R efficiently internalized fluorescein-chelator-PNA-D(Cys-Ser-Lys-Cys) to the cytoplasm, but not with D(Cys-Ala-Ala-Cys). Scintigraphic imaging of MCF7 xenografts in immunocompromised mice revealed that CCND1 and MYC [(99m)Tc]chelator-PNA-D(Cys-Ser-Lys-Cys) probes yielded xenograft. PET imaging with [(64)Cu]chelator-PNA-D(Cys-Ser-Lys-Cys) yielded stronger signals. Scintigraphic imaging of human AsPC1 pancreas cancer xenografts with [(99m)Tc]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys) yielded strong xenograft signals. Stronger xenograft image intensities were obtained by PET imaging of [(64)Cu]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys). MRI required extension of chelator-polydiamidopropanoate dendrimers from the N-termini of the PNA probes to increase the number of contrast paramagnetic gadolinium (III) cations per probe. These results provide a basis for detection of oncogene activity in tissues from outside the body by hybridization with metal-chelator-PNA-peptides that are selectively internalized by cancer cells.

Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs.

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99sTc] chelator-PNA-D (CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.

Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization.

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH.

99mTc-peptide-peptide nucleic acid probes for imaging oncogene mRNAs in tumours.

Imaging oncogene mRNA in tumours would provide a powerful tool for the early detection of occult malignant lesions. The goal was to prepare a chimera consisting of a dodecamer antisense peptide nucleic acid (PNA) specific for c-MYC oncogene overexpressed in human breast cancer cells and a chelating moiety that facilitates quantitative radiolabelling with 99mTc and evaluate it for hybridization and tissue distribution in laboratory animals. The pentapeptide chelator-PNA dodecamer specific for c-MYC mRNA was extended from a solid support by 9-fluorenylmethyloxycarbonyl (Fmoc) coupling. Similarly, a chelator-PNA chimera with four central mismatches was also prepared which served as a control. The chimeras were purified, characterized and evaluated for hybridization to c-MYC mRNA by fluorescent, real-time polymerase chain reaction (RT-PCR). The chimeras were labelled with 99mTc and their tissue distribution was examined in athymic nude mice bearing experimental human breast tumours. 99mTc radiolabelling was quantitative and presented a single peak in reversed phase liquid chromatography. Fluorescent real-time polymerase chain reactions using primer and fluorescent probe sets previously calculated for c-MYC mRNA demonstrated inhibition of reverse transcription by the c-MYC specific chimera as compared to that of the control. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver, and appreciable levels in tumours. These observations suggest that 99mTc-peptide-PNA probes might be useful for imaging gene expression in tumours, and the approach is worthy of further investigation.

[Cleavage of RNA in a hybrid duplex by E. coli ribonuclease H. III. Substrate characteristics of hybrid duplexes of RNA and oligodeoxyribonucleotides containing a bleomycin A5 residue]. / Rasshcheplenie RNK v sostave gibridnykh dupleksov ribonukleazoi H E. coli. III. Substratnye svoistva gibridnykh dupleksov RNK i oligodezoksiribonukleotidov, soderzhashchikh ostatok bleomitsina A5.

The effect of the bleomycin A5 residue linked to four-, eight-, and twelve-mer oligodeoxyribonucleotides on the substrate properties of their tandem and continuous (with or without unmodified octanucleotide effectors) hybrid duplexes was studied using E. coli RNase H. The bleomycin derivatives of oligodeoxyribonucleotides were shown to form hybrid duplexes with practically the same thermostability as those formed by unmodified oligodeoxyribonucleotides. The RNA in the bleomycin-containing hybrid duplexes is cleaved by the E. coli RNase H; however, the initial hydrolysis rate (v0) is 2.6-3.4-fold reduced for the continuous duplexes. In the case of tandem hybrid complexes, the effect of bleomycin on v0 was less pronounced. We hypothesized that steric factors play a key role in the bleomycin inhibition and effectors probably determine the substrate properties of such hybrid complexes. Of all the tandem systems studied, the RNA duplex with the bleomycin-containing tetranucleotide flanked with two effectors displayed the best substrate properties. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

Oligonucleotide treatment of ras-induced tumors in nude mice.

Oligonucleotides have shown an ability to target specific oncogene transcripts and inhibit their expression in cells, but the degree to which sustained treatment can suppress the levels of an oncogenic protein enough to benefit a patient remains to be determined. This question has been studied in several ways. First, the relationship of antisense DNA inhibition to the predicted secondary structure of human H-RAS oncogene mRNA was examined in transformed mouse cells that form solid tumors. Inhibition of H-Ras expression was sequence-specific, dose-dependent, and correlated with inhibition of focus formation. The efficacy of the first intron antisense sequence in reducing H-Ras expression was greater than that of the initiation codon target. Second, H-RAS transformed solid tumor cells were pretreated in vitro with normal oligonucleotides, after which tumor growth from the treated cells was tested in nude mice. The three days of treatment with the first intron antisense DNA reduced H-Ras cellular levels by more than 90% whereas a nonspecific control DNA reduced H-Ras levels by approx 20%. Tumor growth of cells treated with H-RAS antisense oligonucleotide was significantly reduced for up to 14 d following the end of treatment and implantation into the mice, whereas the nonspecific control DNA had no significant effect. Third, H-RAS transformed bladder cancer cells were implanted into nude mice, after which the mice were treated for 31 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with H-RAS 12th codon antisense oligonucleotide was reduced by about 80% throughout the treatment period, reiterating the sustained effect seen in pretreated tumor cells. However, the scrambled phosphorothioate control inhibited tumor growth by about 60%, illustrating some nonspecific inhibition. Fourth, K-RAS transformed pancreatic cancer cells were treated in culture and in nude mice. Inhibition of K-Ras expression with a phosphorothioate oligonucleotide directed against a 5'-UTR sequence was sequence-specific and dose-dependent. K-RAS transformed pancreatic cancer cells were implanted into nude mice, after which the mice were treated for 14 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with K-RAS 5'-UTR antisense oligonucleotide was reduced by about 50% throughout the treatment period, reiterating the sustained effect seen with H-RAS transformed cells. In this case, the sense phosphorothioate control did not inhibit tumor growth, demonstrating that nonspecific inhibition is not a characteristic of all phosphorothioate sequences. The next logical steps include testing oligonucleotide efficacy against other tumor types, toxicological testing in higher species, and clinical trials in human subjects.

Transposon Tn7 gene insertion into an evolutionarily conserved human homolog of Escherichia coli attTn7.

Escherichia coli transposon Tn7 can integrate into its target DNA sequence, attTn7 at the 3' end of glmS, with high specificity and efficiency. Remarkably, the insertional recognition sequence in the E. coli genome displays a high degree of identity with the corresponding region at the 3' end of the corresponding human gene for glutamine-fructose-6-phosphate transaminase (GFPT), located at 2p13. It was therefore of interest to determine whether Tn7 could recognize the corresponding human sequence, and transpose at that site. Strains of E. coli DH5alpha were prepared carrying the tnsA-E genes on one plasmid, and attTn7 or the human equivalent on a second recipient plasmid within the alpha-complementation fragment of the lacZ gene. Each strain was transformed with a donor plasmid carrying a gentamycin resistance gene within the Tn7L and Tn7R cassettes. Restriction mapping and sequence analysis of recipient plasmids isolated from white colonies demonstrated that Tn7 inserted the gentamycin resistance gene both into the E. coli attTn7 sequence, and into its human counterpart. No nonspecific insertion was observed in a control plasmid containing only the lacZ fragment. These results provide a basis to investigate whether TnsA-D proteins can mediate gene insertion into comparably conserved sites in eukaryotic chromosomes.

Comparison of usefulness of estradiol vaginal tablets and estriol vagitories for treatment of vaginal atrophy.

BACKGROUND: Atrophic vaginitis is a common condition. This study compared the usefulness of estradiol vaginal tablets (EVT) and estriol vagitories (EV) in treatment of atrophic vaginitis. METHODS: Ninety-six postmenopausal women with symptoms of atrophic vaginitis were treated for 24 weeks with either EVT or with EV. Patients used the medication daily for the first 2 weeks of the study, and twice-weekly thereafter. RESULTS: Both EVT and EV were effective in treating vaginal atrophy and patients in both treatment groups experienced a significant improvement in vaginal symptoms such as itching, irritation, dryness, and dyspareunia. At the end of the study three (6%) EVT treated women reported leakage and none needed to use sanitary towels. Among the EV treated women 31 (65%) reported leakage and 14 (29%) required sanitary protection. Furthermore, 90% in the EVT group perceived the medication as hygienic compared to 79% in the EV group, and 49% in the EVT group indicated that the product was easy to use compared to 28% in the EV group. Endometrial thickness was increased (1.1 mm with EVT and 0.5 mm on EV) in both treatment groups during the first 2 weeks of the study, but returned to baseline levels when the frequency of drug application was reduced to twice-weekly. CONCLUSIONS: Estradiol vaginal tablets provides an effective alternative to traditional forms of local estrogen therapy.

Increased risk of recurrent venous thromboembolism during hormone replacement therapy--results of the randomized, double-blind, placebo-controlled estrogen in venous thromboembolism trial (EVTET).

Recent observational studies suggest a 2-4 fold increased risk of venous thromboembolism (VTE) in women taking hormone replacement therapy (HRT). The present study was started before publication of these studies, and the aim was to determine if HRT alters the risk of VTE in high risk women. The study was a randomized. double-blind, and placebo-controlled clinical trial with a double-triangular sequential design. Females with previously verified VTE were randomized to 2 mg estradiol plus 1 mg norethisterone acetate, 1 tablet daily (n = 71) or placebo (n = 69). The primary outcome was recurrent deep venous thrombosis (DVT) or pulmonary embolism (PE). Between 1996 and 1998 a total of 140 women were included. The study was terminated prematurely based on the results of circumstantial evidence emerging during the trial. Eight women in the HRT group and one woman in the placebo group developed VTE. The incidence of VTE was 10.7% in the HRT group and 2.3% in the placebo group. In the HRT group, all events happened within 261 days after inclusion. The sequential design did not stop the study, but strongly indicated a difference between the two groups. Our data strongly suggests that women who have previously suffered a VTE have an increased risk of recurrence on HRT. This treatment should therefore be avoided in this patient group if possible. The results also support those of recent epidemiological studies, which also indicate increased risk of VTE in non-selected female populations during HRT.

Antisense c-myc and immunostimulatory oligonucleotide inhibition of tumorigenesis in a murine B-cell lymphoma transplant model.

BACKGROUND: Because the development of drug-resistant cells can lead to relapses in patients with lymphoma treated with chemotherapy, new approaches are needed for effective disease management, such as those targeting the c-MYC proto-oncogene with antisense oligonucleotides. Our goal was to investigate whether antisense c-myc oligonucleotides could prevent tumorigenesis in a B-cell lymphoma model. METHODS: Immunocompetent mice received subcutaneous injections of tumor cells from a transgenic mouse model of Burkitt's lymphoma. For 7 consecutive days, beginning 1 day after tumor cell transplantation, the mice were given either a DNA phosphorothioate oligonucleotide complementary to c-myc codons 1-5 (myc6) or other c-myc-related oligonucleotides at a dose of 0.76 mg per day subcutaneously. Myc protein expression, normalized to beta-actin expression, was measured by western blotting of tumor and splenic proteins. To determine whether tumor inhibition by myc6 could be a result of B-cell activation, we compared the activity of myc6 with that of an immunostimulatory oligonucleotide, mcg. RESULTS: In comparison with control treatments (saline vehicle, scrambled-sequence oligonucleotide, or double-mismatch oligonucleotide), treatment with myc6 delayed tumor onset by 3 days, decreased total tumor mass at sacrifice (i.e., 17 days after tumor cell transplantation) by 40% +/- 16% (mean +/- standard error), and decreased the splenic Myc-to-actin ratio. Inhibition of tumors by myc6 and mcg (both of which share a dACGTT motif) was comparable. Administration of an oligonucleotide sequence complementary to c-myc codons 384-388 (myc55) delayed tumor onset by 5-6 days, decreased total tumor mass at sacrifice by 65% +/- 6%, and reduced the splenic Myc-to-actin ratio to below that produced by myc6. A 14-day treatment regimen of myc55 alternating with mcg completely inhibited tumor formation during the therapeutic schedule. CONCLUSIONS: A combined oligonucleotide regimen, based on antisense c-MYC and immunostimulatory oligonucleotides, should be investigated to increase the number and duration of complete remissions obtained after standard chemotherapy for B-cell lymphoma.

[Poisoning with Jimson weed. Five cases treated with physostigmine]. / Forgiftning med piggeple. Fem tilfeller behandlet med fysostigmin.

During the autumn of 1995, the National Poisons Information Centre was contacted about several cases of poisoning with Jimson weed (Datura stramonium). Five cases are described here. Upon admission to hospital the patients had moderate to severe anticholinergic symptoms, such as mydriasis, sinus tachycardia, agitation, dry mouth, urine retention, fever, hypertension, hallucinations and seizures. Owing to their agitated behaviour, gastrointestinal decontamination was impossible. Repeated doses of physostigmine (2-3 mg) administered intravenously reversed the anticholinergic features without side-effects. In the most severe case, physostigmine was needed for 18 hours (total dose; 25.5 mg). The patients recovered in a day or two, but mydriasis persisted in many cases.

Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insulin-like growth factor 1 for increased cellular uptake.

DNA therapeutics show great potential for gene-specific, nontoxic therapy of a wide variety of diseases. The deoxyribose phosphate backbone of DNA has been modified in a number of ways to improve nuclease stability and cell membrane permeability. Recently, a new DNA derivative with an amide backbone instead of a deoxyribose phosphate backbone, peptide nucleic acid (PNA), has shown tremendous potential as an antisense agent. Although PNAs hybridize very strongly and specifically to RNA and DNA, they are taken up by cells very poorly, limiting their potential as nucleic acid binding agents. To improve cellular uptake of a PNA sequence, it was conjugated to a D-amino acid analog of insulin-like growth factor 1 (IGF1), which binds selectively to the cell surface receptor for insulin-like growth factor 1 (IGF1R). The IGF1 D-peptide analog was assembled on (4-methylbenzhydryl)amine resin, and then the PNA was extended as a continuation of the peptide. The conjugate and control sequences were radiolabeled with 14C or fluorescently labeled with fluorescein isothiocyanate. Cellular uptake of the PNA-peptide conjugate, a control with two alanines in the peptide, and a control PNA without the peptide segment were studied in murine BALB/c 3T3 cells, which express low levels of murine IGF1R, in p6 cells, which are BALB/c 3T3 cells which overexpress a transfected human IGF1R gene, and in human Jurkat cells, which do not express IGF1R, as a negative control. The specific PNA-peptide conjugate displayed much higher uptake than the control PNA, but only in cells expressing IGF1R. This approach may allow cell-specific and tissue-specific application of PNAs as gene-regulating agents in vivo.

Rapid measurement of modified oligonucleotide levels in plasma samples with a fluorophore specific for single-stranded DNA.

Animal studies of therapeutic oligonucleotides require measurement of circulating levels of oligonucleotides by multistep, time-consuming methods. In contrast, addition of a single-stranded DNA binding fluorophore, OliGreen, to oligonucleotides in plasma samples allowed rapid quantitation. Dose-response curves were measured for five different oligonucleotide analogs added to plasma or serum. Phosphorothioate or 3'-amino phosphodiester oligodeoxynucleotides in calf serum reliably exhibited linear, dose-dependent fluorescence at 15-500 nM. The assay was equally sensitive in human and mouse plasma, with a heterogeneous variety of sequences. Oligonucleotides shorter than 10 nucleotides yielded substantially reduced fluorescence. In contrast, 2'-O-methyl oligoribonucleotides, DNA methylphosphonates, and peptide nucleic acids demonstrated little or no fluorescence with OliGreen. Following intravenous injection of a phosphorothioate pentadecamer into mice, fluorescence measurements of plasma phosphorothioate levels displayed a dose-dependent, biexponential decline over a 90 min period. Chronic infusion at 2.5 nmol/hour into mice yielded plasma oligonucleotide values equivalent to 0.1 microM, a value reflecting the contributions of intact and partially degraded strands. Tumor-bearing mouse plasma evidenced high fluorescence values in the absence of oligonucleotide administration, presumably because of elevated intrinsic plasma DNA fragments. Although limited in its ability to differentiate intact from partially degraded strands, OliGreen fluorescence provides a simple, rapid, and sensitive method for measuring circulating levels of phosphorothioate or phosphodiester oligonucleotides in healthy animals or humans.