Biomedical subjects

M J Leibowitz

Publications and source records attributed to M J Leibowitz.

At least 19 recordsLinked to original sources

PubMed2001

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.

Animals↗

PubMed2001

Folding of the group I intron ribozyme from the 26S rRNA gene of Candida albicans.

Preincubation of the group I intron Ca.LSU from Candida albicans at 37 degrees C in the absence of divalent cations results in partial folding of this intron. This is indicated by increased resistance to T1 ribonuclease cleavage of many G residues in most local helices, including P4-P6, as well as the non-local helix P7, where the G binding site is located. These changes correlate with increased gel mobility and activation of catalysis by precursor RNA containing this intron after preincubation. The presence of divalent cations or spermidine during preincubation results in formation of the predicted helices, as indicated by protection of additional G residues. However, addition of these cations during preincubation of the precursor RNA alters its gel mobility and eliminates the preincubation activation of precursor RNA seen in the absence of cations. These results suggest that, in the presence of divalent cations or spermidine, Ca.LSU folds into a more ordered, stable but misfolded conformation that is less able to convert into the catalytically active form than the ribozyme preincubated without cations. These results indicate that, like the group I intron of Tetrahymena, multiple folding pathways exist for Ca.LSU. However, it appears that the role cations play in the multiple folding pathways leading to the catalytically active form may differ between folding of these two group I introns.

Base Sequence↗

PubMed2001

Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide.

PURPOSE: To investigate the potential for delivering large peptides orally by altering their absorptive transport pathways and improving intestinal permeability. The absorptive transport of retro-inverso (R.I.-) K-Tat9 and R.I.-K(biotin)-Tat9, novel peptidic inhibitors of the Tat protein of HIV-1, and their interactions with human SMVT (hSMVT), a high affinity, low capacity transporter, were investigated using Caco-2 and transfected CHO cells. METHODS: Following synthesis on a PAL resin using Fmoc chemistry, the transport of R.I.-K-Tat9 (0.01-25 microM) and R.I.-K(biotin)-Tat9 (0.1-25 microM) was evaluated across Caco-2 cells. The transport and kinetics of biotin, biocytin and desthiobiotin (positive controls for SMVT) were also determined. Uptake of R.I.-K-Tat9 and R.I.K(biotin)-Tat9 (both 0.1-10 microM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. RESULTS: The absorptive transport of R.I.-K-Tat9 was passive, low (Pm approximately 1 x 10(-6) cm/sec) and not concentration dependent. R.I.K(biotin)-Tat9 permeability was 3.2-fold higher than R.I.-K-Tat9 demonstrating active (Ea = 9.1 kcal/mole), concentration dependent and saturable transport (Km = 3.3 microM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 microM) was - 500-fold greater than R.I.-K-Tat9 (at 10 microM). R.I.-K(biotin)-Tat9 transport in Caco-2 and CHO/hSMVT cells was significantly inhibited by known substrates of SMVT including biotin, biocytin, and desthiobiotin. Passive uptake of R.I.-K(biotin)-Tat9 was significantly greater than R.I.-K-Tat9 uptake in CHO/pSPORT cells. CONCLUSIONS: These results demonstrate that the structural modification of R.I.-K-Tat9 to R.I.-K(biotin)-Tat9 altered its intestinal transport pathway resulting in a significant improvement in its absorptive permeability by enhancing nonspecific passive and carrier-mediated uptake by means of SMVT. The specific interactions between R.I.-K(biotin)-Tat9 and SMVT suggest that targeting approaches utilizing transporters such as SMVT may substantially improve the oral delivery of large peptides.

Administration, Oral↗

PubMed2000

Use of high-performance liquid chromatographic fractionation of large RNA molecules in the assay of group I intron ribozyme activity.

Ion-pair reversed-phase high-performance liquid chromatography (HPLC), which has previously been used to fractionate double-stranded DNA molecules, can be applied to single-stranded RNA molecules in the size range of 200-1000 nucleotides. This procedure permits RNA molecules to be separated and recovered rapidly in liquid medium, thereby facilitating recovery. We have used this system to separate an in vitro transcription product containing a group I intron ribozyme from the intermediates and products of the splicing reaction, permitting rapid assay of ribozyme activity without the use of radioactivity.

Chemical Fractionation↗

PubMed2000

Construction of a large plasmid lacking linearizing single restriction sites by simultaneous in vivo recombination and plasmid shuffling in yeast.

Creation of large ( approximately 15 kb) recombinant plasmids can be done in a single step by co-transformation of yeast cells with a partial restriction digest of a plasmid vector and a linear insert whose ends overlap one of the vector restriction sites. This method is used to generate a plasmid expressing the Saccharomyces cerevisiae rRNA genes containing the Ca.LSU group I intron ribozyme from Candida albicans. This plasmid expresses functional rRNA and ribozyme.

Candida albicans↗

PubMed2000

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae.

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.

Antifungal Agents↗

PubMed2000

The [KIL-d] element specifically regulates viral gene expression in yeast.

The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are "healed" in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10(-4)-10(-5) and reappears with variegated phenotypic expression with a frequency of > or =10(-3). This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

Base Sequence↗

PubMed2000

Pentamidine inhibition of group I intron splicing in Candida albicans correlates with growth inhibition.

We previously demonstrated that pentamidine, which has been clinically used against Pneumocystis carinii, inhibits in vitro a group I intron ribozyme from that organism. Another fungal pathogen, Candida albicans, also harbors a group I intron ribozyme (Ca.LSU) in the essential rRNA genes in almost half of the clinical isolates analyzed. To determine whether pentamidine inhibits Ca.LSU in vitro and in cells, phylogenetically closely related intron-containing (4-1) and intronless (62-1) strains were studied. Splicing in vitro of the Ca.LSU group I intron ribozyme was completely inhibited by pentamidine at 200 microM. On rich glucose medium, the intron-containing strain was more sensitive to growth inhibition by pentamidine than was the intronless strain, as measured by disk or broth microdilution assays. On rich glycerol medium, they were equally susceptible to pentamidine. At pentamidine levels selectively inhibiting the intron-containing strain (1 microM) in glucose liquid cultures, inhibition of splicing and rRNA maturation was detected by quantitative reverse transcription-PCR within 1 min with a 10- to 15-fold accumulation of precursor rRNA. No comparable effect was seen in the intronless strain. These results correlate the cellular splicing inhibition of Ca.LSU with the growth inhibition of strain 4-1 harboring Ca.LSU. Broth microdilution assays of 13 Candida strains showed that intron-containing strains were generally more susceptible to pentamidine than the intronless strains. Our data suggest that ribozymes found in pathogenic microorganisms but absent in mammals may be targets for antimicrobial therapy.

Antifungal Agents↗

PubMed1999

Translational effects of peptide antagonists of Tat protein of human immunodeficiency virus type 1.

The Tat (trans-activator of transcription) regulatory protein of human immunodeficiency virus (HIV-1) acts by interacting with the TAR RNA domain of nascent viral transcripts and with cellular proteins to increase viral transcription. In Jurkat-derived HCLE-D36 cells, which are stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene expressed from the TAR-encoding long terminal repeat (LTR) of HIV-1, CAT protein expression is dependent on Tat. The Tat9-K-biotin peptide antagonist of Tat binds specifically to TAR RNA and competes with Tat for binding. In the cellular expression system, Tat9-K-biotin reduces Tat-dependent CAT expression. However, while the Tat antagonist greatly reduces CAT protein production and polysome association of CAT mRNA, it has little effect on CAT mRNA levels, suggesting that the antagonist works at the post-transcriptional level.

Anti-HIV Agents↗

PubMed1998

Inhibition of HIV-1 replication by a Tat RNA-binding domain peptide analog.

The peptidic compound, N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys(biotin)-NH2 (Tat10-biotin), contains the 9-amino acid sequence from the basic domain of the Tat protein responsible for specific interaction with TAR RNA. The cysteine residue provides an attachment site for biotin, which acts as a cellular uptake enhancer. Tat10-biotin binds a fragment of TAR RNA (deltaTAR) avidly and specifically, as measured in an electrophoretic gel shift assay. Tat10-biotin inhibited tat gene-induced expression of a stably transfected chloramphenicol acetyl transferase (CAT) reporter gene linked to the HIV-1 long terminal repeat (LTR) in a model cell assay, but did not inhibit phorbol ester-induced expression of CAT, thereby demonstrating a Tat-dependent mechanism of inhibition. Inhibition of HIV-1 replication after acute infection of MT2 cells was demonstrated by absence of HIV-induced syncytium formation and cytotoxicity, as well as by suppression of reverse transcriptase production. These results suggest that a peptide or peptide mimetic capable of competing with the TAR RNA-binding domain of Tat protein might be useful as a therapeutic agent for AIDS.

Anti-HIV Agents↗

PubMed1998

The [KIL-d] cytoplasmic genetic element of yeast results in epigenetic regulation of viral M double-stranded RNA gene expression.

[KIL-d] is a cytoplasmically inherited genetic trait that causes killer virus-infected cells of Saccharomyces cerevisiae to express the normal killer phenotypes in a/alpha cells, but to show variegated defective killer phenotypes in a or alpha type cells. Mating of [KIL-d] haploids results in "healing" of their phenotypic defects, while meiosis of the resulting diploids results in "resetting" of the variegated, but mitotically stable, defects. We show that [KIL-d] does not reside on the double-stranded RNA genome of killer virus. Thus, the [KIL-d] effect on viral gene expression is epigenetic in nature. Resetting requires nuclear events of meiosis, since [KIL-d] can be cytoplasmically transmitted during cytoduction without causing defects in killer virus expression. Subsequently, mating of these cytoductants followed by meiosis generates spore clones expressing variegated defective phenotypes. Cytoduction of wild-type cytoplasm into a phenotypically defective [KIL-d] haploid fails to heal, nor does simultaneous or sequential expression of both MAT alleles cause healing. Thus, healing is not triggered by the appearance of heterozygosity at the MAT locus, but rather requires the nuclear fusion events which occur during mating. Therefore, [KIL-d] appears to interact with the nucleus in order to exert its effects on gene expression by the killer virus RNA genome.

Base Sequence↗

PubMed1997

Partial sequence of the rRNA operon of Pneumocystis carinii isolates from Puerto Rico.

Several reports indicate geographic variation of isolates of Pneumocystis carinii hominis. We have sequenced the internal transcribed spacer (ITS) region and large subunit Group I intron of rRNA genes from P. carinii DNA obtained from two patients from Puerto Rico. Both can be subclassified as Type II, according to the sequence of the ITS region. A system capable of identifying individual isolates will be an essential tool for epidemiological studies of the organism. The amplification of DNA from fixed tissues may facilitate the processing of a large number of samples.

Base Sequence↗

PubMed1996

Hybridization properties of oligodeoxynucleotide pairs bridged by polyarginine peptides.

The hybridization properties of a series of probes, based on two 9mer oligodeoxynucleotides (designated as I and II) having an appended oligoarginine chain (Rn) to produce peptide-oligonucleotide conjugates or peptide-bridged oligonucleotide pairs (e.g. Rn-I or II-Rn-I), were investigated. For the double-linked probes, we found that the peptide bridge induces the two 9mers to bind complementary single-stranded DNA or RNA targets with substantially enhanced thermal stability. The resulting hybrid with complementary DNA was found to assume a 1:1 complex in the B conformation as judged by UV mixing curves and CD spectroscopy. Complexes of single or double-linked probes with complementary RNA exhibited sensitivity to RNase H digestion. The influence of the identity and chirality of the repeating unit in the bridge, the length of the bridge, the gap size and the salt concentration on the hybridization properties of this new class of oligonucleotide probes was also studied. Our data reveal that these compounds exhibit properties that should prove useful in the development of antisense strategies.

Amino Acid Sequence↗

PubMed1996

Gel shift assay: demonstration of enhanced binding of oligo(delta)-L-ornithine-oligodeoxynucleotide conjugates to complementary DNA and RNA.

An increase in melting temperature for DNA:DNA duplexes had been observed previously (Zhu et al. Antisense Res. Dev. 3:349-356, 1993) when an oligo(delta)ornithine moiety was covalently appended to a short oligodeoxynucleotide. We now report the analysis of duplex formation by electrophoretic gel shift analysis. In the particular example studied, an increase in Tm of 4 degrees C was found to correspond to about a fivefold increase in binding constant. A similar enhancement by the appended cationic peptide was observed when the target strand was RNA. The use of a competitive assay format for avoidance of adsorptive loss at low concentrations (< 10(-7)M) of the oligonucleotide-oligo(delta)ornithine conjugate is presented.

Base Sequence↗

PubMed1995

Use of a polyethylene glycol-peptide conjugate in a competition gel shift assay for screening potential antagonists of HIV-1 Tat protein binding to TAR RNA.

Interference of binding of Tat protein to TAR RNA in HIV-1-infected cells may be a useful therapeutic strategy for AIDS. An electrophoretic assay to screen potential low-molecular-weight (< 2 kDa) Tat antagonists has been established. A radiolabeled TAR RNA fragment (delta TAR) is retarded in mobility when bound by a Tat peptide-polyethylene glycol conjugate (Tat-PEG), which is used in place of the Tat protein. The assay determines the ability of a potential antagonist to compete with Tat-PEG for binding to delta TAR, as measured by interference with the gel shift of delta TAR. To discriminate between specific and nonspecific interactions, the assay is done in the absence or the presence of a 250-fold molar excess of tRNA.

Amino Acid Sequence↗

PubMed1995

Bidirectional effectors of a group I intron ribozyme.

The group I self-splicing introns found in many organisms are competitively inhibited by L-arginine. We have found that L-arginine acts stereoselectively on the Pc1. LSU nuclear group I intron of Pneumocystis carinii, competitively inhibiting the first (cleavage) step of the splicing reaction and stimulating the second (ligation) step. Stimulation of the second step is most clearly demonstrated in reactions whose first step is blocked after 15 min by addition of pentamidine. The guanidine moiety of arginine is required for both effects. L-Canavanine is a more potent inhibitor than L-arginine yet it fails to stimulate. L-Arginine derivatized on its carboxyl group as an amide, ester or peptide is more potent than L-arginine as a stimulator and inhibitor, with di-arginine amide and tri-arginine being the most potent effectors tested. The most potent peptides tested are 10,000 times as effective as L-arginine in inhibiting ribozyme activity, and nearly 400 times as effective as stimulators. Arginine and some of its derivatives apparently bind to site(s) on the ribozyme to alter its conformation to one more active in the second step of splicing while competing with guanosine substrate in the first step. This phenomenon indicates that ribozymes, like protein enzymes, can be inhibited or stimulated by non-substrate low molecular weight compounds, which suggests that such compounds may be developed as pharmacological agents acting on RNA targets.

Amides↗

PubMed1994

Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes.

Small portions of the 18S and the 26S rRNA genes, the entire 5.8S rRNA gene, and internal transcribed spacers ITS1 and ITS2 (located between the 18S and 5.8S rRNA genes and between the 5.8S and 26S rRNA genes, respectively) of Pneumocystis carinii that infect humans were cloned and sequenced. The nucleotide sequences of the 18S, 5.8S, and 26S rRNA genes determined in the study were approximately 90% homologous to those of P. carinii that infect rats, while the sequences of ITS1 and ITS2 of P. carinii from the two different hosts were only 60% homologous. The 18S, 5.8S, and 26S rRNA gene sequences of P. carinii from 15 patient specimens were determined and were found to be identical to each other, whereas the ITS sequences were found to be variable. With the observed sequence variation, it was possible to classify the ITS1 sequences into two types and the ITS2 sequences into three types. P. carinii strains that had the same type of ITS1 sequence could have a different type of ITS2 sequence. On the basis of the sequence types of the two ITS regions, P. carinii from the 15 patients were classified into four groups. P. carinii from three patient specimens were found to contain two different ITS sequence patterns. More surprisingly, one additional specimen was found to have one ITS sequence typical of P. carinii isolates that infect humans and another typical of P. carinii isolates that infect rats. The studies indicate that it is possible to type P. carinii strains on the basis from one patient, suggesting that coinfection with more than one strain of P. carinii may occur in the same patient.

Animals↗

PubMed1993

Variation and in vitro splicing of group I introns in rRNA genes of Pneumocystis carinii.

The sequences of the rRNA genes of Pneumocystis carinii from rat and human sources demonstrate three distinct genotypes based on the group I introns present in these genes. One rat isolate (Pc1) contains such introns in its 16S and 26S rRNA genes, while another rat isolate (Pc2) and a human isolate (Pc3) only contain an intron in the 26S rRNA gene. The four introns all catalyze their own excision from RNA transcripts, and this reaction is inhibited by the anti-pneumocystis drug pentamidine and its analogues. Although they differ in sequence, they are more similar to one another than to group I introns found in other eukaryotic microbes.

Animals↗