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1.
Biotechnol Appl Biochem ; 34(3): 151-9, 2001 12.
Article in English | MEDLINE | ID: mdl-11730482

ABSTRACT

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly-Arg-Arg-Arg-Arg, corresponding to a region of the N-terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N-terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 microM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro-inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 degrees C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.


Subject(s)
Cell Nucleus/metabolism , Lactoferrin/metabolism , Nuclear Localization Signals/metabolism , Peptide Nucleic Acids/metabolism , Amino Acid Motifs , Cell Nucleolus/metabolism , Endocytosis , Fluorescent Dyes/chemistry , Humans , Lactoferrin/chemistry , Molecular Mimicry , Peptide Nucleic Acids/chemistry , Rhodamines/chemistry , Temperature , Tumor Cells, Cultured
2.
Nitric Oxide ; 5(5): 427-31, 2001.
Article in English | MEDLINE | ID: mdl-11587557

ABSTRACT

The presence of Ca(2+)-dependent, heat-stress-activated nitric oxide synthase (NOS) activity in peculiarly shaped, fusiform, and dendritic sponge cells is described for the first time. The NOS activity was evidenced evaluating the conversion of radioactive citrulline from [(14)C]arginine in intact cells from two different species that are phylogenetically unrelated in the class of Demospongiae: Axinella polypoides and Petrosia ficiformis. The production of nitrogen monoxide (NO) was confirmed by electron paramagnetic resonance analysis, and the histochemistry technique of NADPH diaphorase showed a specific localization of NOS activity in a particular network of dendritic cells in the sponge parenchyma. Sponges are the most primitive metazoan group; their evolution dates back 600 million years. The presence of environmental stress-activated NOS activity in these organisms may prove to be the most ancient NO-dependent signaling network in the animal kingdom.


Subject(s)
Calcium/metabolism , Nitric Oxide Synthase/metabolism , Porifera/enzymology , Stress, Physiological/metabolism , Animals , Citrulline/metabolism , Dendritic Cells/metabolism , Electron Spin Resonance Spectroscopy , Hot Temperature , Immunoenzyme Techniques , NADPH Dehydrogenase/metabolism , Nitric Oxide/metabolism
3.
Nat Biotechnol ; 19(3): 231-4, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11231555

ABSTRACT

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.


Subject(s)
Antisense Elements (Genetics)/pharmacology , Down-Regulation/drug effects , Entamoeba histolytica/drug effects , Kanamycin Kinase/metabolism , Peptide Nucleic Acids/pharmacology , Animals , Antisense Elements (Genetics)/genetics , Biomarkers/analysis , Cell Division/drug effects , Entamoeba histolytica/enzymology , Entamoeba histolytica/genetics , Entamoeba histolytica/growth & development , Gentamicins/pharmacology , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Kanamycin Kinase/biosynthesis , Kanamycin Kinase/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Neomycin/metabolism , Peptide Nucleic Acids/genetics , Permeability , Transfection
5.
Cancer Res ; 60(8): 2258-62, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10786693

ABSTRACT

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA translation. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a nuclear localization signal, permits access to the genome. Here we test the ability of dihydrotestosterone (T) covalently linked to PNA to act as a vector for targeting c-myc DNA to prostatic cancer cell nuclei. LNCaP cells, which express the androgen receptor gene, and DU145 cells, in which the androgen receptor gene is silent, offer a model to test this biologically active hormone as a cell-specific vector. T vector was covalently linked to the NH2-terminal position of a PNA complementary to a unique sequence of c-myc oncogene (PNAmyc-T). To localize PNAmyc-T and vector-free PNA within the cells, a rhodamine (R) group was attached at the COOH-terminal position (PNAmyc-R, PNAmyc-TR); cellular uptake was monitored by confocal fluorescence microscopy. PNAmyc-R was detected only in the cytoplasm of both prostatic cell lines, whereas PNAmyc-TR was localized in nuclei as well as in cytoplasm of LNCaP cells. In contrast, PNAmyc-TR uptake in DU145 cells was minimal and exclusively cytoplasmic. In LNCaP cells, MYC protein remained unchanged by exposure to vector-free PNAmyc, whereas a significant and persistent decrease was induced by PNAmyc-T. In DU145 cells, MYC expression was unaltered by PNAmyc with or without the T vector. Our data show that the T vector facilitates cell-selective nuclear localization of PNA and its consequent inhibition of c-myc expression. These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.


Subject(s)
Cell Nucleus/metabolism , Dihydrotestosterone/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Peptide Nucleic Acids/metabolism , Peptide Nucleic Acids/pharmacology , Prostatic Neoplasms/metabolism , Antisense Elements (Genetics)/genetics , Antisense Elements (Genetics)/metabolism , Antisense Elements (Genetics)/pharmacokinetics , Antisense Elements (Genetics)/pharmacology , Biological Transport , Cell Division/drug effects , Cell Survival/drug effects , Cytoplasm/metabolism , Genes, myc/genetics , Genetic Therapy , Humans , Male , Microscopy, Fluorescence , Nuclear Localization Signals , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/pharmacokinetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/therapy , Receptors, Androgen/deficiency , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Substrate Specificity , Tumor Cells, Cultured
6.
New Microbiol ; 22(3): 249-56, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10423744

ABSTRACT

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, two heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from Terra Nova Bay (Ross sea, Antarctica) by using aliphatic and artomatic hydrocarbons as the principal carbon source. These gram-positive, aerobic, cocci-shaped bacteria use a various number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and biphenyl. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both an extracellular and cell-bound surface-active mixture of trehalose lipids which reduced the surface tension of water from 72 mN/m to 32mN/m. This class of glycolipids was found to be produced only by marine rhodococci. The 16S-rRNA gene sequence analysis showed that both strains are members of the G + C rich gram-positive group of the phylum Proteobacteria and was found to be almost identical to that of Rhodococcus fascians DSM 20669. The potential of these strains for in situ bioremediation of contaminated cold marine environment is discussed in the present study.


Subject(s)
Glycolipids/biosynthesis , Hydrocarbons/metabolism , Rhodococcus/isolation & purification , Surface-Active Agents/metabolism , Alkanes/metabolism , Antarctic Regions , Biodegradation, Environmental , Biphenyl Compounds/metabolism , Burkholderia cepacia/drug effects , DNA, Ribosomal/genetics , Glycolipids/pharmacology , Marine Biology , Phylogeny , RNA, Ribosomal, 16S/genetics , Rhodococcus/classification , Rhodococcus/genetics , Rhodococcus/metabolism , Spectrometry, Mass, Fast Atom Bombardment , Surface-Active Agents/pharmacology , Trehalose , Water Microbiology
7.
FEBS Lett ; 451(3): 264-8, 1999 May 28.
Article in English | MEDLINE | ID: mdl-10371202

ABSTRACT

Overexpression of inducible nitric oxide synthase causes the production of high levels of nitric oxide, which, under pathological conditions, leads to immunosuppression and tissue damage. The results recently obtained using peptide nucleic acids, rather than traditional oligonucleotides as antigen and antisense molecules, prompted us to test their efficacy in the regulation of nitric oxide production, thereby overcoming the obstacle of cellular internalization. The cellular permeability of four inducible nitric oxide synthase antisense peptide nucleic acids of different lengths was evaluated. These peptide nucleic acids were covalently linked to a hydrophobic peptide moiety to increase internalization and to a tyrosine to allow selective 125I radiolabelling. Internalization experiments showed a 3-25-fold increase in the membrane permeability of the modified peptide nucleic acids with respect to controls. Inducible nitric oxide synthase inhibition experiments on intact stimulated macrophages RAW 264.7 after passive permeation of the two antisense peptide nucleic acids 3 and 4 demonstrated a significant decrease (43-44%) in protein enzymatic activity with respect to the controls. These data offer a basis for developing a good alternative to conventional drugs directed against inducible nitric oxide synthase overexpression.


Subject(s)
Macrophages/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitric Oxide/metabolism , Peptide Nucleic Acids/metabolism , Peptide Nucleic Acids/pharmacology , Animals , Antisense Elements (Genetics)/genetics , Antisense Elements (Genetics)/metabolism , Antisense Elements (Genetics)/pharmacology , Biological Transport , Cell Line , Mice , Nitric Oxide Synthase Type II , Peptide Nucleic Acids/genetics
8.
AIDS Res Hum Retroviruses ; 15(4): 345-53, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10082118

ABSTRACT

Disseminated infection with Mycobacterium avium complex (MAC) remains the most common serious bacterial infection in patients with advanced AIDS. The organisms that make up this complex are found ubiquitously in the environment, yet rarely cause disseminated disease in nonimmunocompromised human patients; on the contrary, up to 50% of patients with AIDS may ultimately develop the pathology. Hence, therapeutic strategies able to inhibit HIV and Mycobacterium replication are needed. Because of the rapid plasma elimination and toxicity of the most commonly used drugs, daily multiple-drug therapies must often be continued throughout life, frequently causing major side effects and, as a consequence, poor patient compliance. Therefore, alternative strategies that reduce the toxicity of the drugs and allow prolonged application intervals are sorely needed. Since erythrocytes (RBCs) can behave as bioreactors able to convert impermeant prodrugs to membrane-releasable active drugs, new compounds (AZTpEMB, AZTpEMBpAZT, and AZTp2EMB) consisting of both an antiretroviral and an antimicrobial drug were designed and synthesized. Among these, only AZTp2EMB was hydrolyzed by erythrocyte enzymes and could be encapsulated inside RBCs. AZTp2EMB-loaded RBCs slowly released AZT and EMB in culture medium, reducing its concentration by one-half about every 48 hr of incubation at 37 degrees C. Moreover, when AZTp2EMB-loaded erythrocytes were incubated for 6 days in the presence of human macrophages infected with Mycobacterium avium (M. avium) a marked bactericidal effect (>1 log) was observed. Thus, AZTp2EMB-loaded erythrocytes could be used as endogenous bioreactors for AZT and EMB delivery in the treatment of HIV and M. avium infection.


Subject(s)
Anti-HIV Agents/metabolism , Antitubercular Agents/metabolism , Erythrocytes/metabolism , Ethambutol/metabolism , Zidovudine/metabolism , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacology , Bioreactors , Cells, Cultured , Dimerization , Ethambutol/chemical synthesis , Ethambutol/pharmacology , Humans , Molecular Structure , Mycobacterium avium/drug effects , Zidovudine/chemical synthesis , Zidovudine/pharmacology
9.
FEBS Lett ; 426(1): 33-6, 1998 Apr 10.
Article in English | MEDLINE | ID: mdl-9598973

ABSTRACT

Inducible nitric oxide synthase (iNOS) is modulated at the transcriptional level. Overexpression of this protein may result in high levels of nitric oxide leading to tissue damage and immunosuppression. In order to reduce the pathological effects of NO overproduction many efforts have been devoted to the identification of specific inhibitors of iNOS. The discovery of peptide nucleic acids (PNA), a novel class of molecules able to selectively interact with nucleic acids, prompted us to attempt a new way for the regulation of NO production. Here we describe the synthesis, characterization and in vitro effects of a PNA molecule bearing a homopyrimidine sequence complementary to the 5' coding region of murine iNOS mRNA. This PNA shows specific interactions with iNOS mRNA in RNase protection assays and is able to block the synthesis of iNOS protein selectively in a rabbit reticulocyte lysate system. These results strengthen the view of a possible pharmacological application of PNA as a compound able to interfere with a specific enzymatic activity even at low concentrations.


Subject(s)
Nitric Oxide Synthase/genetics , Oligonucleotides, Antisense/chemistry , Animals , Cell-Free System , Mice , Nitric Oxide Synthase Type II , Nucleic Acid Hybridization , Peptides , Protein Biosynthesis/drug effects , RNA, Messenger/chemistry , Rabbits
10.
AIDS Res Hum Retroviruses ; 14(5): 435-44, 1998 Mar 20.
Article in English | MEDLINE | ID: mdl-9546803

ABSTRACT

Human herpesvirus (HSVs) are distributed worldwide and are among the most frequent causes of viral infection in HIV-1-immunocompromised patients. Hence, therapeutic strategies able to inhibit HSV-1 and HIV-1 replication are sorely needed. Until now, the most common therapies against HSV-1 and HIV-1 infectivity have been based on the administration of nucleoside analogs; however, to be active, these antiviral drugs must be converted to their triphosphorylated derivatives by viral and/or cellular kinases. At the cellular level, the main problems involved in the use of such drugs are their limited phosphorylation in some cells (e.g., antiretroviral drugs in macrophages) and the cytotoxic side effects of nucleoside analog triphosphates. To overcome these limitations, a new heterodinucleotide (AZTp2ACV) consisting of both an antiretroviral and an antiherpetic drug, bound by a pyrophosphate bridge, was designed and synthesized. The impermeant AZTp2ACV was encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis by human macrophages. Once inside macrophages, metabolic activation of the drug occurred. The addition of AZTp2ACV-loaded erythrocytes to human macrophages provided effective and almost complete in vitro protection from HIV-1 and HSV-1 replications, respectively. Therefore, AZTp2ACV acts as an efficient antiviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.


Subject(s)
Acyclovir/pharmacology , Antiviral Agents/pharmacology , Erythrocytes , HIV-1/drug effects , Herpesvirus 1, Human/drug effects , Macrophages/virology , Virus Replication/drug effects , Zidovudine/pharmacology , Acyclovir/administration & dosage , Acyclovir/metabolism , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/metabolism , Chlorocebus aethiops , Chromatography, High Pressure Liquid , Cytopathogenic Effect, Viral , Drug Combinations , Erythrocytes/metabolism , HIV-1/growth & development , Herpesvirus 1, Human/growth & development , Humans , Macrophages/physiology , Phagocytosis , Vero Cells , Zidovudine/administration & dosage , Zidovudine/metabolism
11.
J Acquir Immune Defic Syndr Hum Retrovirol ; 17(3): 189-95, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9495216

ABSTRACT

A new antiretroviral drug (azidothymidine homodinucleotide, AZTp2AZT), designed for the protection of macrophages against retroviral infection, was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS) alone and in combination with oral azidothymidine (AZT). C57BL/6 mice were infected with the retroviral complex LP-BM5 and treated for 3 months by weekly administrations of 15 nmol of AZTp2AZT encapsulated into autologous erythrocytes for macrophage protection. AZTp2AZT treatment was found to reduce lymphoadenopathy (48%), splenomegaly (26%), and BM5d proviral DNA content in lymph nodes, spleen, and brain of 37%, 40%, and 36%, respectively, compared with untreated animals. AZT administration in drinking water (0.25 mg/ml) was more effective than administration of AZTp2AZT encapsulated into erythrocytes in reducing lymphoadenopathy, splenomegaly, gammaglobulinemia, and proviral DNA content in lymph nodes, but it caused a reduction in erythrocyte count and hematocrit levels. Although combined treatments do not provide additive responses in the several parameters investigated, they were found to be much more effective in reducing the proviral DNA content in brain (67%) than were monotherapies. Furthermore, no apparent signs of hematotoxicity were observed. Thus, macrophage delivery of antiviral drugs may contribute to brain protection from retroviral infections by mechanisms other than those exerted by oral AZT administration.


Subject(s)
Anti-HIV Agents/therapeutic use , Murine Acquired Immunodeficiency Syndrome/drug therapy , Prodrugs/therapeutic use , Thymine Nucleotides/therapeutic use , Zidovudine/therapeutic use , Animals , Brain/virology , DNA, Viral/analysis , Dideoxynucleotides , Drug Therapy, Combination , Female , Flow Cytometry , Immunoglobulin G/blood , Injections, Intraperitoneal , Lymph Nodes/virology , Lymphatic Diseases/drug therapy , Lymphocyte Activation/drug effects , Lymphocyte Count/drug effects , Macrophages/virology , Mice , Mice, Inbred C57BL , Murine Acquired Immunodeficiency Syndrome/immunology , Murine Acquired Immunodeficiency Syndrome/virology , Prodrugs/administration & dosage , Prodrugs/pharmacology , Spleen/virology , Splenomegaly/drug therapy , Thymine Nucleotides/administration & dosage , Thymine Nucleotides/pharmacology
12.
Biochem Biophys Res Commun ; 236(2): 323-6, 1997 Jul 18.
Article in English | MEDLINE | ID: mdl-9240433

ABSTRACT

Peptide nucleic acid (PNA) molecules are very promising tools for antigene and antisense therapies because of their remarkable refractoriness to degradation in biological fluids. However, very limited information is available on their uptake by potentially target cells and on their intracellular fate. A membrane-diffusable and fluorescence detectable PNA chimera, Phe-Leu-Phe-Leu-(Adenine)3-biotin, was obtained by solid phase peptide synthesis and characterized by combined HPLC and mass spectrometry (MS). This PNA chimera was found to permeate across the membrane of both human erythrocytes and B Namalwa cells much more extensively and rapidly than a control Gly-(Adenine)3-biotin PNA molecule. Fluorescence patterns of internalization were consistent for a diffusion process resulting in the appearance of uniform cytoplasmic distribution of the hydrophobic peptide-PNA chimera in the Namalwa cells. Degradation of the synthesized PNA chimera by cell lysates and to a much slower extent by the intact Namalwa cells was investigated by HPLC-MS analyses of the corresponding methanol extracts . It involved the progressive removal of each of the hydrophobic amino acid residues, while the linkage with the biotin label was completely resistant to cleavage. These results hold promise for the design and synthesis of membrane-permeable PNA sequences suitable for antigene therapies.


Subject(s)
Oligonucleotides/chemistry , Oligopeptides/metabolism , B-Lymphocytes , Biological Transport , Cell Membrane Permeability , Cells, Cultured , Erythrocytes , Humans , Oligopeptides/chemistry , Solubility
13.
Biochem Biophys Res Commun ; 220(1): 20-5, 1996 Mar 07.
Article in English | MEDLINE | ID: mdl-8602844

ABSTRACT

A new Azidothymidine derivative, di-(thymidine-3'- azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphate (AZTp2AZT), was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing. Like in erythrocyte lysates supplemented with 1 mM ATP, intact red cells too were found to convert AZTp2AZT to 3'-Azido-3'-deoxythymidine which was then released linearly in plasma. The major metabolic pathway involved in this conversion was the symmetrical hydrolysis of AZTp2AZT to yield two 3'-Azido-3'- deoxythymidine-5'-phosphate molecules which were then dephosphorylated to 3'-Azido-3'-deoxythymidine. At late times of incubation, also a limited asymmetrical hydrolysis of AZTp2AZT became apparent in the intact erythrocytes, yielding 3'-Azido-3'-deoxythymidine-5'-diphosphate that was then converted to the triphosphorylated derivative. Therefore, erythrocytes loaded with AZTp2AZT act "in vitro" as bioreactors ensuring sustained and potentially useful release of 3'-Azido-3'-deoxythymidine.


Subject(s)
Drug Carriers , Erythrocytes/metabolism , Prodrugs/administration & dosage , Thymine Nucleotides/administration & dosage , Zidovudine/administration & dosage , Dideoxynucleotides , Hemolysis , Humans , In Vitro Techniques , Prodrugs/metabolism , Thymine Nucleotides/metabolism , Zidovudine/metabolism
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