ABSTRACT
Seminal amyloid fibrils are made up of naturally occurring peptide fragments and are key targets for the development of combination microbicides or antiviral drugs. Previously, we reported that the polysulfonic compound ADS-J1 is a potential candidate microbicide that not only inhibits HIV-1 entry, but also seminal fibrils. However, the carcinogenic azo moieties in ADS-J1 preclude its clinical application. Here, we screened several ADS-J1-like analogs and found that the antiparasitic drug suramin most potently inhibited seminal amyloid fibrils. Using various biochemical methods, including Congo red staining, CD analysis, transmission EM, viral infection assays, surface plasmon resonance imaging, and molecular dynamics simulations, we investigated suramin's inhibitory effects and its putative mechanism of action. We found that by forming a multivalent interaction, suramin binds to proteolytic peptides and mature fibrils, thereby inhibiting seminal fibril formation and blocking fibril-mediated enhancement of viral infection. Of note, suramin exhibited potent anti-HIV activities, and combining suramin with several antiretroviral drugs produced synergistic effects against HIV-1 in semen. Suramin also displayed a good safety profile for vaginal application. Moreover, suramin inhibited the semen-derived enhancer of viral infection (SEVI)/semen-mediated enhancement of HIV-1 transcytosis through genital epithelial cells and the subsequent infection of target cells. Collectively, suramin has great potential for further development as a combination microbicide to reduce the spread of the AIDS pandemic by targeting both viral and host factors involved in HIV-1 sexual transmission.
Subject(s)
Amyloid/drug effects , Semen/drug effects , Suramin/pharmacology , Adult , Animals , Anti-HIV Agents/pharmacology , Anti-Retroviral Agents/pharmacology , HIV Infections/metabolism , HIV-1/metabolism , Healthy Volunteers , Humans , Male , Peptide Fragments/metabolism , Peptides/metabolism , Rabbits , Semen/metabolism , Suramin/metabolismABSTRACT
To investigate the enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes. Six compounds, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa were selected as model drugs. Permeation studies were performed using excised cornea of rabbits by a Franz-type diffusion apparatus. The safety of borneol was assessed on the basis of corneal hydration level and Draize eye test. The application of 0.2% borneol to the cornea increased the apparent permeability coefficient by 1.82-(P<0.05), 2.49-(P<0.05), 4.18-(P<0.05) and 1.11-fold (not significant) for rhodamine B, sodium-fluorescein, FITC-dextrans of 4 and 10 kDa, respectively. No significant permeability enhancement of FITC dextrans of 10, 20 and 40 kDa with borneol was found compared to control. The permeability coefficient enhanced by 0.2% borneol was linear correlated to the molecular weight of model drugs (R(2)=0.9976). With the 0.05%, 0.1% and 0.2% borneol application, the corneal hydration values were <83% and Draize scores were <4. Borneol may improve the transcorneal penetration of both hydrophilic and lipophilic compounds without causing toxic reactions, especially hydrophilic ones. Furthermore, 0.2% borneol can enhance the permeation of hydrophilic compounds with molecular weight ≤4 kDa. Hence, borneol can be considered as a safe and effective penetration enhancer for ocular drug administration.
Subject(s)
Adjuvants, Pharmaceutic/pharmacology , Camphanes/pharmacology , Cornea/metabolism , Animals , Dextrans/chemistry , Dextrans/metabolism , Fluorescein/chemistry , Fluorescein/metabolism , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Hydrophobic and Hydrophilic Interactions , Permeability/drug effects , Rabbits , Rhodamines/chemistry , Rhodamines/metabolismABSTRACT
OBJECTIVE: To construct two recombinant lentiviral vectors carrying mouse NMNAT1 gene and RNAi targeting NMNAT1. METHODS: According to GenBank, the full-length cDNA sequence of mouse NMNAT1, an interfering sequence targeting NMNAT1 and a negative sequence were designed, synthesized and inserted into plasmid pLenti6 lentiviral vector. The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells. The virus titer was tested by qPCR methods. After infection of Hela cells with these lentiviruses, the expression of NMNAT1 was detected by qPCR and Western blot. RESULTS: All the recombinant plasmids were confirmed by sequencing. The titer of virus was over 2 X10(8) TU/mL. Hela cells infected with lentiviral vector carrying full length NMNAT1 gene successfully expressed high-level NMNAT1. The expression of NMNAT1 reduced to less than 30% after delivery of lentiviral vector carrying RNAi sequence. CONCLUSION: The lentiviral vectors carrying full length NMNAT1 gene and RNAi sequence targeting NMNAT1 have been successfully constructed.
Subject(s)
Lentivirus/genetics , Nicotinamide-Nucleotide Adenylyltransferase/genetics , RNA Interference , Animals , Gene Expression , Genetic Vectors , HeLa Cells , Humans , Mice , Plasmids/genetics , RNA, Small Interfering/genetics , TransfectionABSTRACT
BACKGROUND: Wallerian degeneration is a self-destructive process of axonal degeneration that occurs after an axonal injury or during neurodegenerative disorders such as Parkinson's or Alzheimer's disease. Recent studies have found that the activity of the nicotinamide adenine dinucleotide (NAD) synthase enzyme, nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) can affect the rate of Wallerian degeneration in mice and drosophila. NMNAT1 protects neurons and axons from degeneration. However, the role of NMNAT1 in neurons of central nervous system is still not well understood. METHODS: We set up the culture of primary mouse neurons in vitro and manipulated the expression level of NMNAT1 by RNA interference and gene overexpression methods. Using electroporation transfection we can up-regulate or down-regulate NMNAT1 in cultured mouse dendrites and axons and study the neuronal morphogenesis by immunocytochemistry. In all functional assays, FK-866 (CAS 658084-64-1), a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase was used as a pharmacological and positive control. RESULTS: Our results showed that knocking down NMNAT1 by RNA interference led to a marked decrease in dendrite outgrowth and branching and a significant decrease in axon growth and branching in developing cortical neurons in vitro. CONCLUSIONS: These findings reveal a novel role for NMNAT1 in the morphogenesis of developing cortical neurons, which indicate that the loss of function of NMNAT1 may contribute to different neurodegenerative disorders in central nervous system.
Subject(s)
Axons/metabolism , Dendrites/metabolism , Neurons/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Animals , Blotting, Western , Cells, Cultured , Immunohistochemistry , Mice , Morphogenesis/genetics , Morphogenesis/physiology , Neurons/cytology , Nicotinamide-Nucleotide Adenylyltransferase/geneticsABSTRACT
OBJECTIVE: To construct and identification of a lentiviral vector for RNA interference (RNAi) targeting STUB1 gene. METHODS: A pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting STUB1 gene was designed, synthesized and inserted into linearized pMagic 4.0 vector. The recombinant plasmid was identified by double restriction digestion with Age I/EcoR I and DNA sequencing. RESULT: PCR and DNA sequencing showed that the shRNA sequence was successfully inserted into pMagic 4.0 vector. The pMagic 4.0 vector was successfully packaged into lentivirus particles. CONCLUSION: A lentiviral shRNA expression vector and particles targeting STUB1 gene has been successfully constructed for the further study of the STUB1 gene.
Subject(s)
Genetic Vectors , Lentivirus/genetics , RNA Interference , Ubiquitin-Protein Ligases/genetics , Gene Targeting , RNA, Small Interfering/geneticsABSTRACT
AIM: To explore the method of injecting the BDNF gene lentiviral vector in rat hippocampus and detecting its expression. METHODS: SD rats were randomly divided into four groups: sham operated group (SH), normal saline group (NS), GFP gene lentiviral vector injection group (GFP) and BDNF lentiviral vector injection group (BDNF).The expression of BDNF was detected by Western blot, RT-PCR and situ hybridization (ISH) at day 7, 14 and 30 after the injection. RESULTS: BDNF Gene lentiviral vector can up-regulated the expression of BDNF in rat hippocampus. The result of RT- PCR and Western blot detection show that the level of mRNA and the protein level was remarkably increased. The result of in-situ hybridization showed the stable expression and distribution of BDNF. CONCLUSION: Successfully established the method of injecting BDNF gene lentiviral vector in rat hippocampus and detected the high level of BDNF expression following with the time, which lays the basis for its application in the treatment of the neurodamaged disease.
Subject(s)
Brain-Derived Neurotrophic Factor/genetics , Hippocampus/metabolism , Lentivirus/genetics , Animals , Blotting, Western , Genetic Vectors , In Situ Hybridization , Male , Rats , Rats, Sprague-DawleyABSTRACT
The localization of the voltage-gated calcium channel (VGCC) alpha2 and the voltage-gated sodium channel (VGSC) alpha subunits was immunohistochemically investigated in chicken spinal motoneurons. Approximately 83% and 46% of spinal motoneurons were positive for VGCCalpha2 and VGSCalpha subunits, respectively. Almost all VGSCalpha subunit-positive motoneurons exhibited the VGCCalpha2 subunit immunoreactivity. There were different patterns in occurrence, intensity or nuclear/cytoplasmic stainability of the VGCCalpha2 and VGSCalpha subunits among the motoneurons. This study presents the first cellular morphological evidence for the VGCCalpha2 and VGSCalpha subunits in spinal motoneurons, postulating that the heterogeneous expression of VGCCalpha2 and VGSCalpha subunits in the motoneurons may reflect various motor activities.