Biomedical subjects

C Subasinghe

Publications and source records attributed to C Subasinghe.

12 recordsLinked to original sources

PubMed2001

[Is there justification for varicose vein surgery with local anesthesia in successive procedures? Prospective study of 5,000 crossectomy/stripping operations].

UNLABELLED: In a prospective study we examined the complications of 5,000 operations (n = 876 as outpatient; ligations of the SFJ/SPJ +/- stripping or recurrence operation of the SFJ/SPJ), that were carried out under local anaesthesia in a successive procedure. We registered only the in-hospital complications or those occurring during the first postoperative week of outpatient procedure: Mortality, deep venous thrombosis, pulmonary embolism, major vascular injuries, major nerval injuries, anaesthesia complications. The proof and/or exclusion of complications were done by clinical investigations with the parameters: Mortality, vascular and/or nerval injuries, anaesthesia complications as well as pulmonary embolism. With the parameter deep venous thrombosis n = 2,495 operations were examined by color duplex scan in the region of the sapheno-femoral (popliteal) junction before patients were discharged from hospital/ambulatory therapy. In the other patients the diagnosis was done according to clinical parameters. RESULTS: In the investigation period two complications occurred at the nerval-system after difficult ligation of the SPJ: a) Paresis concerning mainly the peroneal branch of the N. ischiadicus with regeneration after nine months. b) Lesion of the N. tibialis with paresis of the M. abductor digiti minimi that was unchanged after seven months. CONCLUSION: Varicose vein surgery under local anaesthesia as a successive procedure represents a very safe treatment option. A comparison with literature data from prospective or retrospective studies is not possible, because of missing studies and/or unclear information or definition problems. It would be desirable if such studies could be performed.

Adult↗

PubMed1996

Prenatal detection of de novo inversion of chromosome (2) (p13q11.2) and postnatal follow-up.

We report the first case of an apparent de novo pericentric inversion of chromosome 2 at the breakpoints p13q11.2 that was detected prenatally. Follow-up performed over 4 years showed phenotypic abnormalities including minor craniofacial dysmorphism, hypotonia, hearing loss, gustatory flushing syndrome, and severe developmental delays. The literature on chromosome 2 inversion is reviewed.

Abnormalities, Multiple↗

PubMed1992

A comparison of gag, pol and rev antisense oligodeoxynucleotides as inhibitors of HIV-1.

Sequences from the gag, pol and rev regions of the RF strain of HIV-1 (HIV-1RF) were chosen as targets for antisense phosphorothioate oligodeoxynucleotides (S-oligos). These sequences were the p18/p24 junction in gag, the active site of HIV protease in pol; a sequence from the first exon of the rev gene and S-oligodeoxycytidylic acid controls. Compounds were tested against HIV-1 in both acutely and chronically infected cells. The results show that these phosphorothioate analogues tested in acutely infected cells were active in the 0.1-2 microM range, were dependent on chain length but had no sequence specificity. To study the mechanism of action, the time of addition of S-oligos to acutely infected cells was delayed for up to 48 h post-infection. It was found that antiviral activity was lost when compounds were added to the cultures later than 10 h post-infection. With chronically infected cells only the antisense rev sequence showed activity at 30 microM and neither of the gag or pol antisense sequences has a significant effect on HIV replication at 50 microM. These results are consistent with previous in vitro studies which demonstrate that antisense S-oligodeoxynucleotides have several modes of action.

Antiviral Agents↗

PubMed1990

Antisense-mediated inhibition of BCL2 protooncogene expression and leukemic cell growth and survival: comparisons of phosphodiester and phosphorothioate oligodeoxynucleotides.

Antisense oligodeoxynucleotides specific for sequences in mRNAs from the B-cell lymphoma/leukemia-2 (BCL2) gene were used to inhibit the growth in culture of a human leukemia cell line, 697. Normal phosphodiester (PO) and nuclease-resistant phosphorothioate (PS) oligodeoxynucleotides were compared with regard to specificity, potency, and kinetics. Both PO and PS antisense BCL2 oligodeoxynucleotides were specific inhibitors of cellular proliferation, since sense versions of these synthetic DNAs were inactive at similar concentrations. Specificity was further confirmed by quantitative immunofluorescence studies, showing that PO and PS antisense BCL2 oligodeoxynucleotides (when used at appropriate concentrations) reduced levels of BCL2 protein without influencing expression of HLA-DR and other control antigens. The onset of inhibition by PO oligodeoxynucleotides was faster, with reductions in cell numbers occurring within 1 day of addition to cultures, in contrast to phosphorothioates, which were ineffective until 3-4 days. Phosphorothioates were more potent that phosphodiesters, however, with half-maximal inhibition of leukemic cell growth occurring at concentrations 5-10 times lower. As expected from previous studies demonstrating the importance of BCL2 for regulating lymphoid cell survival, BCL2 antisense oligodeoxynucleotides also led to 697 leukemic cell death through sequence-specific mechanisms, with reductions in cellular viability generally lagging the inhibitory effects on cellular growth by about 2 days. Taken together, these data indicate that PO and PS oligodeoxynucleotides targeted against the human BCL2 protooncogene can be sequence-specific inhibitors of leukemic cell growth and survival.

Cell Division↗

PubMed1990

Preparation of 35S-labeled polyphosphorothioate oligodeoxyribonucleotides by use of hydrogen phosphonate chemistry.

The title compounds were chemically synthesized as their 5'-dimethoxytrityl derivatives by base-catalyzed reaction of 35S-enriched elemental sulfur with support-bound hydrogen phosphonate oligomer. This was derived from adamantane carbonyl chloride-activated coupling of nucleotide hydrogen phosphonate monomers, and similarly activated capping with isopropyl phosphite. A convenient, disposable, reversed-phase cartridge was utilized to purify and isolate the 5'-dimethoxytrityl derivative for subsequent in situ detritylation and elution of the final product. The specific activity obtained for the title compounds was ca. 10(7) cpm/mumols-eq P(O)S-. The procedure should be readily adaptable to appropriate syntheses of other P-S containing analogs of DNA and RNA.

Base Sequence↗

PubMed1989

Phosphoroselenoate oligodeoxynucleotides: synthesis, physico-chemical characterization, anti-sense inhibitory properties and anti-HIV activity.

Oligodeoxynucleotides with a phosphorus atom in which one of the non-bridging oxygen atoms is substituted by selenium were prepared and investigated with respect to their antisense properties. A general synthesis of phosphoroselenoate analogs of oligonucleotides is described using potassium selenocyanate as the selenium donor. The compounds, characterized by 31P NMR, were shown to decompose to phosphate with a half-life of ca. 30 days. Melting temperatures of duplexes between poly(rA) or poly(rI) with oligo(dT) and oligo(dC), respectively, indicate diminished hybridization capability of phosphoroselenoate oligomers relative to both the unmodified phosphodiester oligomers and the phosphorothioate congeners. A phosphoroselenoate 17-mer is a sequence specific inhibitor of rabbit beta-globin synthesis in wheat germ extract and in injected Xenopus oocytes. In contrast phosphoroselenoate analogs are potent non-sequence specific inhibitors in rabbit reticulocyte lysate. In vitro HIV assays were carried out on a phosphoroselenoate sequence and compared with a phosphorothioate analogue that has previously been shown to exhibit anti-HIV activity (Matsukura et al., Proc. Natl. Acad. Sci. (1987) 84, 7706-7710). The phosphoroselenoate was somewhat less active, and was much more toxic to the cells.

Animals↗

PubMed1989

Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides.

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides.

Animals↗

PubMed1989

Oligodeoxynucleotide analogs with 5'-linked anthraquinone.

We report here the synthesis of novel 5'-linked oligodeoxynucleotides, both normal phosphodiester and phosphorothioate analogs, in which a covalently attached group at the 5'-terminus is an anthraquinone. These compounds represent a new class of antisense compounds in which the base sequence of the oligodeoxynucleotide serves to deliver a nuclease-resistant reactive drug-like molecule to a cellular target nucleic acid (mRNA or DNA).

Anthraquinones↗

PubMed1989

Characterization of oligonucleotide transport into living cells.

Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptor-mediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.

Acridine Orange↗

PubMed1989

Phosphorothioate oligodeoxynucleotides are potent sequence nonspecific inhibitors of de novo infection by HIV.

Phosphorothioate homo-oligodeoxynucleotides have recently been found to protect ATH-8 cells against the cytopathic effect of de novo infection by HIV. The effect is dose and chain-length dependent, with a maximum effect seen for 21-28-mers. We have now synthesized a series of phosphorothioate oligomers with mixed-based sequences and found that all of them have a dose-dependent cytoprotective effect that is maximal at an oligomer concentration of about 1-2 microM. The least effective sequences contain only A or T, and the most effective sequences have 40% GC content or greater. The results also confirm the length effect, namely that 21-mers are more cytoprotective than 14-mers.

Antiviral Agents↗

PubMed1988

Phosphorothioate and normal oligodeoxyribonucleotides with 5'-linked acridine: characterization and preliminary kinetics of cellular uptake.

Certain phosphorothioate oligodeoxynucleotide (S-oligo) analogs, unlike their normal congeners, have been found to exhibit significant anti-HIV activity [Matsukura et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7706-7710]. Here we report melting temperatures (Tm) of a series of S-oligos compared with those of the corresponding normal oligomers. The Tm's for AT base pairs of S-oligos are significantly depressed relative to normal oligos, while GC-containing S-oligos show much less Tm depression. The Tm's of S-dT oligomers with poly(rA) are reduced relative to the duplexes with normal dA oligomers. These results provide a rational basis for the S-d(CG) sequences as anti-message inhibitors of gene expression. We also describe an automated synthesis of 5'-acridine linked oligothymidylates using phosphoramidite-linked acridine. During this synthesis we noted the replacement of thiophenol for the 6-chloro substituent on acridine. We have measured the Tm's of the compounds with 3 and 5 methylene groups linked to normal and phosphorothioate dTn (with n = 3-40) on duplex formation with the equivalent dAn, and have found small increases of Tm for the 5-methylene-linked acridine derivative. We have monitored the uptake of these fluorescently labeled oligos into HL60 cells, and found that the shorter oligos are more rapidly taken up than the longer, and the normal oligos faster than the S-oligos. The temperature dependence of the cellular uptake suggests an energy-dependent process, and a possible membrane receptor for oligos. These results have significance for the potential use of such compounds as inhibitors of gene expression.

Acridines↗

PubMed1988

Physicochemical properties of phosphorothioate oligodeoxynucleotides.

We have recently shown that phosphorothioate (PS) oligodeoxynucleotide (ODN) analogs, unlike their normal congeners, exhibit significant anti-HIV activity (Matsukura et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7706-7710). We now report the syntheses, melting temperatures (Tm), and nuclease susceptibilities of a series of phosphorothioate ODN analogs. These include all-PS duplexes, duplexes with one normal chain and the other chain either all-PS, or end-capped with several PS groups at both 3' and 5' ends. The DNase susceptibilities of the S-ODNs are much less than the normal phosphodiesters, but by contrast duplexes of poly-rA with S-dT40 are much more susceptible to RNase H digestion. The Tm's for AT base pairs of S-ODNs are significantly depressed relative to normals, while GC base pairs show much less Tm depression. The Tm's of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers. These results have significance for the biological properties of these analogs as anti-message inhibitors of gene expression, and provide a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.

Chemical Phenomena↗