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Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery in vivocould affect penetration or interception in biological environments. The aim of this study was to assess the feasibility of cyclo-(D-Trp-Tyr) PNTs with two ARs as carriers for oral pMBP-bcl-xL-hRlucdelivery to the spinal cord to treat spinal cord injury (SCI). We evaluated the biodistribution of oligodendrocyte (OLG)-specific myelin basic protein gene promoter-driven antiapoptotic DNA (pMBP-bcl-xL) to the brain and spinal cord delivered with cyclo-(D-Trp-Tyr) PNTs with large (L) and small (S) PNTs with two ARs. After complex formation, the length, width, and AR of the L-PNTs/DNA were 77.86 ± 3.30, 6.51 ± 0.28, and 13.75 ± 7.29 μm, respectively, and the length and width of the S-PNTs/DNA were 1.17 ± 0.52 and 0.17 ± 0.05 μm, respectively, giving an AR of 7.12 ± 3.17 as detected by scanning electron microscopy. Each of these three parameters exhibited significant differences (p< 0.05) between L-PNTs/DNA and S-PNTs/DNA. However, there were no significant differences (p> 0.05) between the L-PNTs and S-PNTs for either their DNA encapsulation efficiency (29.72 ± 14.19 and 34.31 ± 16.78%, respectively) or loading efficiency (5.15 ± 2.58 and 5.95 ± 2.91%). The results of the in vitroanalysis showed that the S-PNT/DNA complexes had a significantly higher DNA release rate and DNA permeation in the duodenum than the L-PNT/DNA complexes. Using Cy5 and TM-rhodamine to individually and chemically conjugate the PNTs with plasmid DNA, we observed, using laser confocal microscopy, that the PNTs and DNA colocalized in complexes. We further confirmed the complexation between DNA and the PNTs using fluorescence resonance energy transfer (FRET). Data from an in vivoimaging system (IVIS) showed that there was no significant difference (p> 0.05) in PNT distribution between L-PNTs/DNA and S-PNTs/DNA within 4 h. However, the S-PNT/DNA group had a significantly higher DNA distribution (p< 0.05) in several organs, including the ilium, heart, lungs, spleen, kidneys, testes, brain, and spinal cord. Finally, we determined the bcl-xLprotein expression levels in the brain and spinal cord regions for the L-PNT/DNA and S-PNT/DNA complex formulations. These results suggested that either L-PNTs or S-PNTs may be used as potential carriers for oral gene delivery to treat SCI.

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO–PPO–PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitroand in vivoon the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO–PPO–PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB–DNA). In addition, the in vitrocorneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB–DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB–DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO–PPO–PEO polymers as a vehicle for drug delivery.

The aliphatic polyesters PLLA (poly L-lactic acid) and PBSA (poly (butylene succinate)-co-(butylene adipate)) were used to fabricate nonwoven-type scaffolds with sub-micrometer fiber diameters via an electrospinning method. The electrospun membrane surfaces were further coated with either γ-PGA (poly-γ-glutamic acid) or collagen through physical bonding. These specialized surfaces were employed as peripheral nerve conduits to regulate cell adhesion and cell proliferation in vitro of the neuron cell line PC12. Furthermore, the expression of neuron growth-related (GAP-43, MAP-2) and apoptosis-related genes (Bcl-xL, Bcl-2, Bax, Fas) was evaluated for PC12 cells cultured on the electrospun membranes in response to NGF (nerve growth factor) treatment. No significant differences in apoptosis-related gene expression were observed as a function of membrane type. The GAP-43 and MAP-2 genes were highly expressed on the PLLA samples with NGF but performed variably on the PBSA samples depending on the type of coating material. Our results suggest that electrospun PLLA membranes can serve as proper matrices for neuron outgrowth and that electrospun PBSA membranes are suitable surfaces for PC12 cell attachment and proliferation.

The feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as oral gene delivery carriers was investigated in nude mice with eight 40 μg doses of pCMV-lacZin 2 days at 3 h intervals. The association between DNA and PNTs, the DNase I stability of PNTs-associated DNA, and in vitropermeability of DNA were estimated. The results showed that the cyclo-(d-Trp-Tyr) PNTs self-associated at concentrations above 0.01 mg/mL. Plasmid DNA associated with PNTs with a binding constant of 3.2 × 108M–1calculated by a fluorescence quenching assay. PNTs were able to protect DNA from DNase I, acid, and bile digestion for 50 min, 60 min, and 180 min, respectively. The in vitroduodenal apparent permeability coefficient of pCMV-lacZcalculated from a steady state flux was increased from 49.2 ± 21.6 × 10–10cm/s of naked DNA to 395.6 ± 142.2 × 10–10cm/s of pCMV-lacZ/PNT formulation. The permeation of pCMV-lacZformulated with PNTs was found in an energy-dependent process. Furthermore, β-galatosidase (β-Gal) activity in tissues was quantitatively assessed using chlorophenol red-β-d-galactopyranoside (CPRG) and was significantly increased by 41% in the kidneys at 48 h and by 49, 63, and 46% in the stomach, duodenum, and liver, respectively, at 72 h after the first dose of oral delivery of pCMV-lacZ/PNT formulation. The organs with β-Gal activity were confirmed for the presence of pCMV-lacZDNA with Southern blotting analysis and intracellular tracing the TM-rhodamine-labeled DNA and the presence of mRNA by reverse transcription-real time quantitative PCR (RT-qPCR). Another plasmid (pCMV-hRluc) encoding Renilla reniformisluciferase was used to confirm the results. An increased hRlucmRNA and luciferase in stomach, duodenum, liver, and kidney were detected by RT-qPCR, ex vivobioluminescence imaging, luciferase activity quantification, and immunostaining, respectively.

This study attempts to investigate the transdermal permeability, the bioavailability and gene expression of plasmid formulated with nonionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM). Dynamic light scattering (DLS) and atomic force microscopy (AFM) were used to analyze the PM formulated pCMV-LacZ (P/PM) containing the gene for β-galactosidase (β-Gal) driven by cytomegalovirus early promoter. Franz diffusion cell was used for in vitrotransdermal permeability analysis. Real-time PCR was used to quantify the permeated plasmid in vitroand in vivo. β-Gal activity assay was performed to evaluate transgene expression in vivo. The size of P/PM was ∼50 nm with round shape. PM significantly enhanced the in vitrotransdermal permeability of plasmid in a direction- and temperature-dependent manner. Following transdermal application of P/PM, higher area under the curve (AUCP/PM: 98.34 h·ng/mL) and longer half-life of plasmid were detected compared with that of plasmid alone (AUCP: 10.12 h·ng/mL). Additionally, the β-Gal activity was significantly increased in skin, stomach, brain and spinal cord at both 48 and 72 h after P/PM application and in testis and spleen at 72 h postapplication. In conclusion, PM formulation enhanced the permeation of plasmid through skin into blood circulation, increasing its absorption and the transgene expression in various tissues.

The use of biodegradable synthetic polyesters as peripheral nerve conduits and the “contact guidance” effect of nano-sized fibrous matrices on neurite outgrowth have been reported; however, no literature exists regarding how these matrices regulate neurite growth-related gene expression. Such information would be useful in the evaluation of the biological applicability of various materials. In this study, two biodegradable synthetic polyesters (PLLA and PBSA) were employed to fabricate a series of micro/nano-fibrous membranes that were used as culture matrices for the in vitro growth of PC12 cells in response to nerve growth factor (NGF). Cell adhesion, proliferation, and differentiation were analyzed in addition to neurite outgrowth- and apoptosis-related gene expression. Our results show that cells grown on electrospun PBSA membranes proliferate to a greater extent than cells cultured on tissue culture polystyrene (TCPS) and other prepared membranes. Compared to TCPS and PBSA, growth on an electrospun PLLA membrane triggers higher GAP43 and MAP2 gene expression in PC12 cells. No significant effect of membrane type on apoptosis-related gene expression was observed. These results indicate that electrospun nano-fibrous PLLA mats are more suitable for use as nerve conduit matrices than conventional non/micro-fibrous PLLA or PBSA membranes.

From the screening of 21 microbial strains, Absidia pseudocylindrosporaATCC 24169 and Aspergillus nigerBCRC 32720 were found to reproducibly transform isosteviol lactam (4α-carboxy-13α-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam) (3) into various compounds. Preparative-scale transformation of 3with Abs. pseudocylindrosporayielded two new hydroxylated compounds (4and 5), with conservation of the lactam ring. Preparative-scale transformation of 3with Asp. nigerafforded seven new compounds, 6and 9–14, together with the known compounds 7and 8. A single-crystal X-ray diffraction experiment confirmed the structure of 14. The suppressive effects of compounds 1–14on the lipopolysaccharide-induced expression of the inducible nitric oxide synthase gene in RAW 264.7 macrophages were examined by a reverse-transcription real-time PCR analysis. With the exception of 7, all other compounds significantly reduced levels of iNOS mRNA relative to control cells, which were induced by LPS alone. Compounds 2, 3, and 5were similar in activity to dexamethasone, while 9was more potent.

Two filamentous fungi, Cunninghamella bainieriATCC 9244 and Aspergillus nigerBCRC 32720, were used to investigate the biotransformation of isosteviol lactone (4α-carboxy-13α-hydroxy-13,16- seco-ent-19-norbeyeran-16-oic acid 13,16-lactone) ( 2), which was derived by reacting isosteviol ( ent-16-oxobeyeran-19-oic acid) ( 1) with m-chloroperbenzoic acid. Incubation of 2with C. bainieriafforded metabolites 3– 6, which involved isomerization, hydroxylation, and ring cleavage reactions followed by oxidation and selective O-methylation. Incubation of 2with A. nigerafforded mono-, di-, and trihydroxylated metabolites 3, 4, and 7– 12. The structures of 3– 12were elucidated on the basis of spectroscopic analyses, and structures 3, 4, and 6were confirmed by X-ray crystallographic studies. Compounds 2– 6, 8– 10, and 12were assayed as androgen agonists using an ARE (androgen response element)-mediated luciferase reporter gene assay. Compounds 3, 6, and 10were significantly active, with 6showing more activity than testosterone.

Preparative-scale fermentation of isosteviol ( ent-16-oxobeyeran-19-oic acid) ( 1) with Mucor recurvatusMR 36, Absidia pseudocylindrosporaATCC 24169, and Aspergillus nigerBCRC 32720 afforded nine known metabolites ( 2, 3, 5– 10, and 14) and nine new metabolites ( 4, 11– 13, and 15– 19). The reactions involved stereoselective introduction of OH groups at positions C-1, -6, -7, -9, -11, -12, -15, and -17 as well as further ketonization at the C-1 and C-7 positions. The structures of the metabolites were established on the basis of 1D and 2D NMR and IR supported by HRFABMS. GRE (glucocorticoid response element)- and ARE (androgen response element)-mediated luciferase reporter gene assays were used to screen for the biological activities of 1and its metabolites. Compounds 7, 13, 16, and 18showed significantly enhanced GRE-mediated luciferase activity, but at levels less than that induced by either methylprednisolone or dexamethasone. On the other hand, compounds 3, 4, 12, 13, 14, and 18showed significant effects on ARE-mediated luciferase activity; in particular, 3, 12, 14, and 18were more active than testosterone.

Eight new ent-beyerane metabolites, 5−8, 12, and 14−16, and four new ent-kaurane metabolites, 3, 10, 11, and 13, together with two known metabolites, 4and 9, were isolated from the microbial transformations of steviol-16,17-epoxide using Streptomyces griseusATCC 10137 and Cunninghamella bainieriATCC 9244. The structures of the metabolites were characterized by IR, HRFABMS, and 1D and 2D NMR data. In addition, a GRE (glucocorticoid response element)-mediated luciferase reporter assay was used to initially screen for the biological activity of the 11 metabolites and stevioside. Steviol (1), steviol-16,17-epoxide (2), ent-11,13,16,17-tetrahydroxykauran-19-oic acid (3), ent-17-hydroxy-16-ketobeyeran-19-oic acid (4), ent-9,13-dihydroxy-16,17-epoxykauran-19-oic acid (10), ent-9,17-dihydroxy-16-ketobeyeran-19-oic acid (12), ent-1,17-dihydroxy-16-ketobeyeran-19-oic acid (14), and stevioside showed significant effects; in particular, stevioside showed almost equal potency as dexamethasone.

The main aim of this study was to investigate the feasibility of using nonionic polymeric micelles of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) as a carrier for oral DNA delivery in vivo. The size and appearance of DNA/PEO-PPO-PEO polymeric micelles were examined, respectively, by dynamic light scattering and atomic force microscopy, and their ζ potential was measured. Expression of the delivered lacZ gene in various tissues of nude mice was assessed qualitatively by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside staining of sections and quantitatively by measuring enzyme activity in tissue extracts, using the substrate of β-galactosidase, chlorophenol red-β-D-galactopyranoside. In addition, the types of cells expressing the lacZ gene in the duodenum were identified by histological analysis. DNA/PEO-PPO-PEO polymeric micelles are a single population of rounded micelles with a mean diameter of 170 nm and a ζ potential of -4.3 mV. Duodenal penetration of DNA/PEO-PPO-PEO polymeric micelles was evaluated in vitro by calculating the apparent permeability coefficient. The results showed a dose-independent penetration rate of (5.75 ± 0.37) × 10-5 cm/sec at low DNA concentrations (0.026-0.26 μg/μl), but a decrease to (2.89 ± 0.37) × 10-5 cm/sec at a concentration of 1.3 μg/μl. Furthermore, when 10 mM RGD peptide or 10 mM EDTA was administered before and concurrent with the administration of DNA/PEO-PPO-PEO polymeric micelles, transport was inhibited ([0.95 ± 0.57] × 10-5 cm/sec) by blocking endocytosis or enhanced ([29.8 ± 5.7] × 10-5 cm/sec) by opening tight junctions, respectively. After oral administration of six doses at 8-hr intervals, the highest expression of transferred gene lacZ was seen 48 hr after administration of the first dose, with gene expression detected in the villi, crypts, and goblet cells of the duodenum and in the crypt cells of the stomach. Reporter gene activity was seen in the duodenum, stomach, and liver. Activity was also seen in the brain and testis when mice were administered 10 mM EDTA before and concurrent with DNA/PEO-PPO-PEO polymeric micelle administration. lacZ mRNA was detected in these five organs and in the blood by reverse transcription-polymerase chain reaction. Taken together, these results show efficient, stable gene transfer can be achieved in mice by oral delivery of PEO-PPO-PEO polymeric micelles.

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg↓Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were ∼31–32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0°C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.

CYP21 gene encodes the steroid 21‐hydroxylase (P450c21) that is involved in steroidogenesis in the adrenal cortex. Mutations occurring on CYP21 which convert it to the neighboring pseudogene, CYP21P, are found in patients with congenital adrenal hyperplasia (CAH), an autosomal recessive disease. We previously reported that the CYP21P pseudogene had lower transcription activity when compared with the active CYP21 gene. The sequences determining the basal transcription activity of the human CYP21 gene are located within the 166 bp region upstream from the transcriptional start site. Within this region, only 4 nucleotides are different between the active CYP21 and the CYP21P pseudogene; they are located at the –117, –104, –101 and –94 positions from the start site of the gene. Here, we report that the CYP21 gene‐specific G nucleotide sequence at the –104 position is required for its basal transcription activity driven by the native TATA box of the human CYP21 gene. When this single nucleotide is changed to the CYP21P sequence, the basal transcription activity decreases by ∼80% in transient transfection assay. In addition, our data from gel retardation assay show that this sequence is also critical for interaction with nuclear proteins from adrenal cells. These results therefore suggest that the single G sequence of the human CYP21 gene is crucial for the expression of its basal transcription activity, and this may be influenced by the interaction with specific nuclear proteins from the adrenal gland.

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169-173. Substitutions of Ile-171 and −172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower Vmaxvalue. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.