Biomedical subjects

J Temsamani

Publications and source records attributed to J Temsamani.

At least 19 recordsLinked to original sources

PubMed2005

Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties.

One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 microg/mL puromycin for the first 2 days of culture or 3 microg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 x 10(-6) cm/s) and a high electrical resistance (up to 500 Omega x cm(2)). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations.

Animals↗

PubMed2001

Translocation of the pAntp peptide and its amphipathic analogue AP-2AL.

The pAntp peptide, corresponding to the third helix of the homeodomain of the Antennapedia protein, enters by a receptor-independent process into eukaryotic cells. The interaction between the pAntp peptide and the phospholipid matrix of the plasma membrane seems to be the first step involved in the translocation mechanism. However, the mechanism by which the peptide translocates through the cell membrane is still not well established. We have investigated the translocation ability of pAntp through a protein-free phospholipid membrane in comparison with a more amphipathic analogue. We show by fluorescence spectroscopy, circular dichroism, NMR spectroscopy, and molecular modeling that pAntp is not sufficiently helically amphipathic to cross a phospholipid membrane of a model system. Due to its primary sequence related to its DNA binding ability in the Antennapedia homeodomain-DNA complex, the pAntp peptide does not belong to the amphipathic alpha-helical peptide family whose members are able to translocate by pore formation.

Amino Acid Sequence↗

PubMed2001

Physico-chemical requirements for cellular uptake of pAntp peptide. Role of lipid-binding affinity.

The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptor-independent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide-lipid interaction in this mechanism and the physico-chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala-Scan mapping. The peptides were labeled with a fluorescent group (7-nitrobenz-2-oxo-1,3-diazol-4-yl-; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process. To further investigate which are the physico-chemical requirements for phospholipid-binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane-mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.

Alanine↗

PubMed2001

Doxorubicin-peptide conjugates overcome multidrug resistance.

A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein (P-gp), which is capable of lowering intracellular drug concentrations. To overcome this problem, we tested the capability of two peptide vectors that are able to cross cellular membranes to deliver doxorubicin in P-gp-expressing cells. The antitumor effect of peptide-conjugated doxorubicin was tested in human erythroleukemic (K562/ ADR) resistant cells. The conjugate showed potent dose-dependent inhibition of cell growth against K562/ADR cells as compared with doxorubicin alone. Doxorubicin exhibited IC50 concentrations of 65 microM in the resistant cells, whereas vectorized doxorubicin was more effective with IC50 concentrations of 3 microM. After treatment of the resistant cells with verapamil, the intracellular levels of doxorubicin were markedly increased and consequent cytotoxicity was improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement in the cell uptake nor in the cytotoxic effect of the conjugated doxorubicin, indicating that the conjugate bypasses the P-gp. Finally, we show by the in situ brain perfusion method in P-gp-deficient and competent mice that vectorized doxorubicin bypasses the P-gp present at the luminal site of the blood-brain barrier. These results indicate that vectorization of doxorubicin with peptide vectors is effective in overcoming multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Vector-mediated drug delivery to the brain.

As a consequence of the growing ageing population, many neurodegenerative diseases, cancer and infections of the brain will become more prevalent. Despite major advances in neuroscience, many potential therapeutic agents are denied access to the central nervous system (CNS) because of the existence of the blood-brain barrier (BBB). This barrier is formed by the endothelial cells of the brain capillaries and its primary characteristic is the impermeability of the capillary wall due to the presence of complex tight junctions and a low endocytic activity. The BBB behaves as a continuous lipid bilayer and prevents the passage of polar and lipid-insoluble substances. The BBB is, therefore, the major obstacle to drugs that are potentially useful for combating diseases affecting the CNS. Extensive efforts have been made to develop CNS drug delivery strategies in order to enhance delivery of therapeutic molecules across the BBB. The current challenge is to develop drug-delivery strategies that will allow the passage of therapeutic drugs through the BBB in a safe and effective manner. This review focuses specifically on the strategies developed to enhance drug delivery across the BBB with an emphasis on the vector-mediated strategy.

Animals↗

PubMed2001

Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy: saturation kinetics and specificity.

Doxorubicin delivery to the brain is often restricted because of the poor transport of this therapeutic molecule through the blood-brain barrier (BBB). To overcome this problem, we have recently developed a technology, Pep:trans, based on short natural-derived peptides that are able to cross efficiently the BBB without compromising its integrity. In this study, we have used the in situ mouse brain perfusion method to evaluate the brain uptake of free and vectorized doxorubicin. Doxorubicin was coupled covalently to small peptide vectors: L-SynB1 (18 amino acids), L-SynB3 (10 amino acids), and its enantio form D-SynB3. We first confirmed the very low brain uptake of free radiolabeled doxorubicin, which is most likely due to the efflux activity of the P-glycoprotein at the level of the BBB. Vectorization with either L-SynB1, L-SynB3, or D-SynB3 significantly increased the brain uptake of doxorubicin (about 30-fold). We also investigated the mechanism of transport of vectorized doxorubicin. We show that vectorized doxorubicin uses a saturable transport mechanism to cross the BBB. The effect of poly(L-lysine) and protamine, endocytosis inhibitors, on the transport across the brain was also investigated. Both inhibitors reduced the brain uptake of vectorized doxorubicin in a dose-dependent manner. These studies indicate that the transport of vectorized doxorubicin appears to occur via an adsorptive-mediated endocytosis.

Algorithms↗

PubMed2000

Brain drug delivery technologies: novel approaches for transporting therapeutics.

The blood-brain barrier (BBB) denies many therapeutic agents access to brain tumours and other diseases of the central nervous system (CNS). Despite remarkable advances in our understanding of the mechanisms involved in the development of the brain diseases and the actions of neuroactive agents, drug delivery to the brain remains a challenge. For more than 20 years, extensive efforts have been made to enhance delivery of therapeutic molecules across vascular barriers of the CNS. The current challenge is to develop drug-delivery strategies that will allow the passage of drug molecules through the BBB in a safe and effective manner, and this review will provide an insight into some of the strategies developed to enhance drug delivery across the BBB.

Journal Article↗

PubMed2000

New advances in the transport of doxorubicin through the blood-brain barrier by a peptide vector-mediated strategy.

Many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain barrier (BBB). To overcome this problem, we have developed a novel method in which short, naturally derived peptides (16-18 amino acids) cross the cellular membranes of the BBB with high efficiency and without compromising its integrity. The antineoplastic agent doxorubicin (dox) was coupled covalently to two peptides, D-penetratin and SynB1. The ability of dox to cross the BBB was studied using an in situ rat brain perfusion technique and also by i.v. injection in mice. In the brain perfusion studies, we first confirmed the very low brain uptake of free radiolabeled dox because of the efflux activity of P-glycoprotein at the BBB. By contrast, we have demonstrated that when dox is coupled to either the D-penetratin or SynB1 vectors, its uptake was increased by a factor of 6, suggesting that the vectorized dox bypasses P-glycoprotein. Moreover, using a capillary depletion method, we have shown that vectorization of dox led to a 20-fold increase in the amount of dox transported into brain parenchyma. Intravenous administration of vectorized dox at a dose of 2.5 mg/kg in mice led to a significant increase in brain dox concentrations during the first 30 min of postadministration, compared with free dox. Additionally, vectorization led to a significant decrease of dox concentrations in the heart. In summary, our results establish that the two peptide vectors used in this study enhance the delivery of dox across the BBB.

Animals↗

PubMed1998

Cellular distribution of phosphorothioate oligonucleotide following intravenous administration in mice.

Oligonucleotides are promising therapeutic agents for the prevention or treatment of a variety of diseases. The therapeutic potential of oligonucleotide therapy depends greatly on the bioavailability of oligonucleotides to their target cells and organs. We previously reported the pharmacokinetics and distribution of phosphorothioate oligonucleotide in mice using [35S]-labeled oligonucleotide ([35S]-oligo). To extend this study, we administered 30 mg/kg of fluorescent-labeled oligonucleotide (FITC-oligo) to mice and examined oligonucleotide distribution by measuring the fluorescence intensity in various cells and tissues using flow cytometry. Following FITC-oligo administration, fluorescence was detected in all the tissues examined. In terms of the fluorescent intensity, accumulation was greatest in liver and kidney, intermediate in spleen and bone marrow, and very low in peripheral blood mononuclear cells (PBMC). At 4 hours after administration, the level of oligonucleotide uptake in PBMC, spleen lymphocytes, and bone marrow cells revealed the following pattern: monocytes/macrophages > B cells > T cells. Confocal microscopy detected intracellular fluorescence in PBMC prepared under the same conditions as those for flow cytometry. These studies provide a rationale for designing cell targets for antisense therapeutics.

Animals↗

PubMed1998

[Antisense oligonucleotides: a new therapeutic approach].

The use of antisense oligonucleotides as therapeutic agents has generated considerable enthusiasm in the research and medical community. Oligonucleotides inhibit gene expression by binding to their target nucleic acid with high specificity and selectivity. The field of antisense technology has progressed enormously. Major progress has been accomplished in the synthesis and manufacturing of modified oligonucleotides. Numerous studies have demonstrated the ability of antisense oligonucleotides to modulate gene expression, in such diverse fields as infectious diseases, cancer, and inflammation. More than a dozen of clinical trials using antisense oligonucleotides have been initiated during the last three years or so. The insights gained through these ongoing clinical trials has opened the pathway to the design of more advanced chemistries which have improved safety profile and efficacy.

Animals↗

PubMed1997

In vivo metabolic profile of a phosphorothioate oligodeoxyribonucleotide.

Antisense phosphorothioate oligodeoxyribonucleotides (PS oligonucleotides) have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. Following administration in vivo, PS oligonucleotides are rapidly cleared from the plasma and distributed to various organs. However, the manner in which administered oligonucleotides are metabolized in plasma and tissues is poorly understood. In this study, a 25-mer PS oligonucleotide (GEM91) complementary to the gag gene mRNA of the human immunodeficiency virus (HIV-1) was administered to mice through intravenous injections to investigate its metabolism. The PS oligonucleotide was extracted from plasma at 1 hour postadministration and from kidney and liver at 24 hours postadministration. After extraction, the PS oligonucleotide and its metabolites were tailed with dA and annealed to a dT-tailed plasmid. The recombinant plasmid was ligated and used to transform competent bacteria. The region of interest containing the PS oligonucleotide was then sequenced. Our results show that degradation of the PS oligonucleotide in plasma was primarily from the 3'-end. However, in kidney and liver, degradation was primarily from the 3'-end, but a large proportion of the PS oligonucleotide was degraded from the 5'-end as well. We also studied the metabolism of PS oligonucleotide in plasma after 2-hour intravenous infusion in HIV-infected patients. The degradation of the PS oligonucleotide in plasma was primarily from the 3'-end. This study is important in understanding the metabolism of antisense PS oligonucleotide in vivo in general but also provides guidance for designing second-generation antisense oligonucleotides with improved stability and safety profile.

Animals↗

PubMed1997

Pattern and kinetics of cytokine production following administration of phosphorothioate oligonucleotides in mice.

Phosphorothioate oligonucleotides with certain sequences or structure motifs can stimulate the immune system. We administered to mice a 27-mer phosphorothioate oligonucleotide (sequence 5'-TCG TCG CTG TCT CCG CTT CTT CTT GCC-3'), which has previously been shown to cause splenomegaly and hypergamma-globulinemia on in vivo administration in mice, and studied the pattern and kinetics of cytokine production at both the splenic mRNA and serum protein levels. Following i.p. administration of 50 mg/kg of oligonucleotide, significant increases in the splenic mRNA levels of IL-6, IL-12p40, IL-1 beta, and IL-1Ra and serum levels of IL-6, IL-12, MIP-1 beta, and MCP-1 were observed. In contrast, no significant differences in splenic mRNA levels of IL-2, IL-4, IL-5, IL-9, IL-13, IL-15, IFN-gamma, or MIF or serum levels of IL-2, IL-4, IL-5, IL-10, IFN-gamma, or GM-CSF were detected. The induction of IL-12 secretion was dependent on the sequence and dose of the oligonucleotides. One oligonucleotide (sequence 5'-GAG AAC GCT CGA CCT TCG AT-3') induced a high level of IL-12 secretion even at 5 mg/kg, whereas another oligonucleotide (sequence 5'-CTC TGC CAC CCA TCT CTC TCC TTC T-3') did not induce significant IL-12 secretion even at 50 mg/kg. IL-12 secretion induced by various doses of oligonucleotide has the same kinetics but differs in magnitude. These studies show a distinct pattern and kinetics of cytokine production following oligonucleotide administration and further demonstrate that cytokine induction is not a general property of phosphorothioate oligonucleotides but is dependent on the sequence and dose of the oligonucleotides.

Animals↗

PubMed1997

Antisense approach for the treatment of cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common viral opportunistic infection in patients suffering with acquired immunodeficiency virus (AIDS). HCMV is a systemic infection that may infect several sites in the body, including the retina, gastrointestinal tract, lungs, liver, and central nervous system. Retinitis is the most frequent manifestation of HCMV infection, occurring in 15-40% of all patients. HCMV is progressive and destroys the retina, eventually leading to blindness. Although, there are several drugs available to treat this disease, they are often of limited efficacy and have significant side-effects. Antisense oligonucleotides represent a novel alternative to the currently available drugs. Due to their high affinity and specificity to target the HCMV RNAs, interest in antisense technology to treat HCMV infections has been intense during the past few years. Two antisense drugs are currently in clinical trials, ISIS 2922 (Formivirsen) and GEM 132.

Journal Article↗

PubMed1997

Antisense oligonucleotides: a new therapeutic approach.

The use of antisense oligonucleotides as therapeutic agents has heralded a new field of genetic pharmacology. Oligonucleotides are relatively easy to design and display increased affinity and selectivity for their nucleic acid targets compared with traditional drugs. However, the development of antisense therapy has not been as simple as was first believed; many critical issues had to be addressed. The first generation of oligonucleotides investigated as drug candidates were phosphorothioate oligonucleotides. Several animal experiments have provided evidence that antisense oligonucleotides can inhibit gene expression of disease-associated proteins. These promising studies led to the advancement of these compounds into clinical trials in such diverse fields as infectious diseases, cancer and inflammation. The insights gained through ongoing clinical trials has opened the pathway to the design of second-generation oligonucleotides which have an improved safety profile and efficacy.

Animals↗

PubMed1996

Modulation of oligonucleotide-induced immune stimulation by cyclodextrin analogs.

Some phosphorothioate oligonucleotides have been shown previously to stimulate cell proliferation and immunoglobulin production. In the current study, we examined the effects of cyclodextrin analogs as immunomodulatory agents for oligonucleotide-induced immune stimulation, both in vitro and in vivo. Incubation of splenocytes with a 27-mer phosphorothioate oligonucleotide that induces immune stimulation increased cell proliferation as measured by [3H]thymidine incorporation, whereas treatment of splenocytes with the phosphorothioate oligonucleotide complexed to cyclodextrin analogs markedly reduced oligonucleotide-induced cell proliferation. Similarly, administration of the 27-mer phosphorothioate oligonucleotide into mice resulted in splenomegaly and an increase in IgM production 48 hr post-administration. Administration of the oligonucleotide along with cyclodextrin analogs resulted in a significant suppression of splenomegaly and IgM response. Such suppression was dependent on the concentration of cyclodextrin analogs and was observed with various other immune stimulatory phosphorothioate oligonucleotide sequences. Administration of cyclodextrin analogs alone had no effect on splenomegaly or immune stimulation.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1996

The C-group heterogeneous nuclear ribonucleoprotein proteins bind to the 5' stem-loop of the U2 small nuclear ribonucleoprotein particle.

The C-group heterogeneous nuclear ribonucleoprotein (hnRNP) proteins bind to nascent pre-messenger RNA. In vitro studies have indicated that the C hnRNP proteins bind particularly strongly to the intron polypyrimidine tract of pre-mRNA and may be important for pre-mRNA splicing. In addition, there is evidence that the interaction of the C hnRNP proteins with pre-mRNA is facilitated by the U1 and U2 small nuclear RNPs (snRNPs). In the present study, we have uncovered another feature of the C hnRNP proteins that may provide a unifying framework for these previous observations; the C hnRNP proteins bind to the 5' stem-loop of the U2 snRNP. This was detected by incubating human 32P-labeled U2 snRNP in micrococcal nuclease-treated HeLa nuclear extracts, followed by UV-mediated protein-RNA cross-linking, which revealed that C hnRNP proteins were cross-linked to 32P-nucleotides in the U2 snRNP. In similar experiments, no cross-linking of C hnRNP proteins to 32P-labeled U1 or U4 snRNPs was observed. The observed cross-linking of C hnRNP proteins to U2 snRNP was efficiently competed by excess U2 RNA and by poly(U) but not by poly(A). No competition was observed with an RNA molecule comprising U2 nucleotides 105-189, indicating that the C hnRNP protein interactive regions of U2 RNA reside solely in the 5' half of the molecule. Oligodeoxynucleotide-mediated RNase H cleavage experiments revealed that a 5' region of U2 RNA including nucleotides 15-28 is essential for the observed C hnRNP protein cross-linking. C hnRNP protein cross-linking to U2 snRNP was efficiently competed by a mini-RNA corresponding to the first 29 nucleotides of U2 RNA, whereas no competition was observed with a variant of this mini-RNA in which the UUUU loop of stem-loop I was mutationally configured into a single-stranded RNA by replacing the stem with non-pairing nucleotides. Competition experiments with another mutant mini-U2 RNA in which the UUUU loop was replaced by AAAA indicated that both the UUUU loop and the stem are important for C hnRNP protein cross-linking, a finding consistent with other recent data on the RNA sequence specificity of C hnRNP protein binding.

Binding, Competitive↗

PubMed1996

Effect of different chemically modified oligodeoxynucleotides on immune stimulation.

Based on previous studies that certain oligonucleotides can stimulate cell proliferation and immunoglobulin production, this study was carried out to establish the relationship between the stimulatory effect and the chemical modification of the oligonucleotide. First, the effects of oligonucleotide and analogs on immune stimulation were studied in vitro using murine splenic lymphocytes. Our results show that cell proliferation and immunoglobulin production (IgG and IgM) depend on the sequence and the chemical modification of the oligonucleotide. Phosphorothioate oligodeoxynucleotides displayed a greater stimulatory effect than partially modified phosphorothioate oligonucleotides. Second, we studied the effects of these chemically modified oligonucleotides after injection in mice. Massive splenomegaly and stimulation of cell proliferation were observed with some phosphorothioate oligonucleotides. These effects were minimized markedly by chimeric and hybrid oligonucleotides. We also demonstrate that in vitro the effects of oligonucleotides on murine lymphocytes were unaffected by T cell depletion, suggesting that oligonucleotides exert their effects mainly on the B cells.

Adjuvants, Immunologic↗

PubMed1996

Enzymatic labeling of nucleic acids.

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.

DNA↗