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Biomedical subjects

K Leong

Publications and source records attributed to K Leong.

10 recordsLinked to original sources
PubMed1991

In vitro release of hydrophobic drugs from polyanhydride disks.

We have evaluated the feasibility of using polyanhydride disks containing hydrophobic antiproliferative agents for controlled drug release after glaucoma filtration surgery. Taxol and VP-16 were the most potent inhibitors of fibroblast proliferation tested, with ID50s of 3 ng/mL and 200 ng/mL, respectively. In vitro release of taxol occurred at concentrations exceeding its ID50 for at least 100 days; VP-16 was released for 31 days. The media into which the drugs were released were able to inhibit fibroblast proliferation in vitro, indicating that the bioactivity of the drugs withstood incorporation into, and release from, the polyanhydride. Polyanhydride disks containing taxol and VP-16 merit testing in animal models of glaucoma filtration surgery.

Alkaloids↗
PubMed1990

High-level transcription from the adenovirus major late promoter requires downstream binding sites for late-phase-specific factors.

The adenovirus major late promoter (MLP) is active during both the early and late phases of infection. During the early phase the activity of the MLP is similar to those of the other early viral promoters, but during the late phase the rate of transcription from the MLP becomes much greater by comparison. We report here that sequence-specific binding proteins are induced during the late phase which interact with three regions in the first intron of the MLP transcription unit from positions +37 to +68, +80 to +105, and +105 to +125 relative to the transcription initiation site. To measure the significance of these binding sites for transcription during the late phase, we constructed MLP-beta-globin fusions and substituted them for early region 3 in adenovirus recombinants. Deletion of the binding sites caused significant reductions in the rate of transcription, specifically during the late phase of infection. Deletion of all three sites reduced the rate of transcription 25- to 50-fold and the accumulation of cytoplasmic MLP-beta-globin RNA 200-fold. These results indicate that the high rate of transcription from the MLP during the late phase of infection results from the interaction of virus-induced transcription factors with three binding sites in the first intron of the major late transcription unit.

Adenoviruses, Human↗
PubMed1989

Ultrasound-enhanced polymer degradation and release of incorporated substances.

The effect of ultrasound on the degradation of polymers and the release rate of incorporated molecules within those polymers was examined. Up to 5-fold reversible increases in degradation rate and up to 20-fold reversible increases in release rate of incorporated molecules were observed with biodegradable polyanhydrides, polyglycolides, and polylactides. Up to 10-fold reversible increases in release rate of incorporated molecules within nonerodible ethylene/vinyl acetate copolymer were also observed. The release rate increased in proportion to the intensity of ultrasound. Temperature and mixing were relatively unimportant in effecting enhanced polymer degradation, whereas cavitation appeared to play a significant role. Increased release rates were also observed when ultrasound was applied to biodegradable polymers implanted in rats. Histological examination revealed no differences between normal rat skin and rat skin that had been exposed to ultrasonic radiation for 1 hr. With further study, ultrasound may prove useful as a way of externally regulating release rates from polymers in a variety of situations where on-demand release is required.

Animals↗
PubMed1988

Polyanhydride microsphere formulation by solvent extraction.

A novel process based on solvent extraction was developed to produce drug-loaded polyanhydride microspheres for controlled-release applications. The technique consists of adding a chloroform solution of polyanhydride and drug into a stirred silicone oil phase containing suspended droplets of surfactant. No chemical reaction, heating, nor contact with water was required in this process. The microspheres produced were capable of releasing various dyes for prolonged periods of time.

Anhydrides↗
PubMed1988

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.

Adenovirus Early Proteins↗
PubMed1986

Adenovirus early region 1A protein increases the number of template molecules transcribed in cell-free extracts.

Protein encoded by adenovirus early region 1A (E1A) stimulates transcription from adenovirus promoters in vivo. Here we show that this effect can be observed in vitro. In a run-off transcription assay from the adenovirus serotype 2 (Ad2) major late promoter, extracts prepared 20 hr postinfection were 5-15 times more active than mock-infected-cell extracts prepared in parallel. Similar results were observed for in vitro transcription from the protein IX and E3 adenovirus promoters, whereas a 2-fold increase was observed for the human beta-globin promoter. The increased activities of infected-cell extracts did not depend on the expression of viral late proteins or the small E1A-encoded proteins but did require expression of the large E1A protein. These results are consistent with the large E1A protein stimulating transcription in vitro as it does in vivo. By limiting in vitro transcription to one initiation per template, we found that the higher activity of an infected-cell extract was due to an increase in the number of templates transcribed. These results suggest that the large E1A protein either increases the number of active transcription factors in infected cells or facilitates the interaction of cellular transcription factors with promoter DNA.

Adenovirus Early Proteins↗
PubMed1985

The expression of the SV40 early region in the transformed human cell line SV80.

The one complete copy of the SV40 early region present in human cells of the transformed line SV80 carries a duplication of the 5' portion of the early region, including its transcriptional control region and splicing signals (W. Gish and M. Botchan, personal communication). Novel SV40-specific RNA species of sufficiently large size, 3.8 and 4.2 kb, to be expressed from the duplicated early transcriptional control region were detected in SV80 cytoplasmic and nuclear RNA preparations by blot hybridization. The results of transcription in a cell-free system of a plasmid, pSV80-04, representing this SV80 cell SV40 DNA integrate (W. Gish and M. Botchan, personal communication) and of nuclease protection experiments with end-labelled pSV80-04 DNA fragments support the conclusion that the duplicated early sequences are transcribed in SV80 cells. It has also been established that the duplicated early splicing signals are functional in SV80 cells. These results are discussed in relation to the large amounts of SV40 early mRNA and T-antigen synthesized in cells of the SV80 line.

Antigens, Viral, Tumor↗
PubMed1985

Primer-dependent eukaryotic RNA polymerase capable of accurate transcription from the adenovirus major late promoter in a reconstituted system.

A sensitive assay for detection of eukaryotic RNA polymerase II has been developed. This assay depends on the ability of polymerase II to elongate a small RNA primer, oligo(U), hybridized to a single-stranded homopolymeric DNA template, poly(dA). The poly(dA).oligo(U)-dependent RNA polymerase II from calf thymus has been purified approximately 10,000-fold using this assay. The purified enzyme contains four polypeptides of apparent Mr 180,000, 140,000, 24,000, and 16,000 and is fully active in accurate initiation of transcription from the adenovirus major late promoter in the presence of transcription factors from HeLa cells. The poly(dA).oligo(U)-dependent RNA polymerase activity can be detected in crude cell extracts from a variety of tissue culture cells and appears to be largely due to polymerase II, since 90-95% of this activity is inhibited by alpha-amanitin at a concentration of 1 microgram/ml.

Adenoviruses, Human↗
PubMed1978

Inhibition of translation by poliovirus: inactivation of a specific initiation factor.

Translation of vesicular stomatitis virus (VSV) mRNA, like host mRNA translation, is inhibited in cells infected with poliovirus. To study the mechanism of poliovirus-induced inhibition of protein synthesis, we prepared extracts from poliovirus-infected and uninfected HeLa cells. Poliovirus mRNA was translated in lysates from both infected and uninfected cells, while VSV mRNA was translated only in the lysate from uninfected cells. Addition of purified translation initiation factors to the extract from infected cells showed that one factor, eIF-4B, could restore VSV mRNA translation in the infected lysate, but did not increase poliovirus mRNA translation. Further experiments involving translation of VSV mRNA in mixed extracts from poliovirus-infected and uninfected cells showed (i) that there was not an excess of an inhibitor of VSV mRNA translation in the infected lysate, but (ii) that an acitivity that caused a slow inactivation of eIF-4B was present in the infected lysate. Inactivation of eIF-4B appears to be the mechanism by which poliovirus infection causes a selective inhibition of translation.

Cell-Free System↗