34 research outputs found
Full-length TNC and TNCIII1â5 cross-competition ELISA.
<p>Testing of binding of full length TNC to a selection of growth factors and cross-competition binding assay of TNC with TNCIII1â5. (A) Binding of TNC (10 nM) to PDGF-BB, BDNF, VEGF-A165, FGF-2, and BMP-2. (B) An increasing dose of full length TNC (0 to 50 nM) resulted in higher binding levels of TNC to PDGF-BB (black). No specific binding to BSA (grey) was observed. (CâG) Cross-competition assay between full length TNC and TNCIII1â5 (10 nM) to bind selected growth factors: PDGF-BB (C), BDNF (D), VEGF-A165 (E), FGF-2 (F), and BMP-2 (G). The TNC concentration varied from 0.1 nM to 50 nM (nâ=â3, mean±SD), with 100% representing binding of 10 nM TNCIII1â5 without the presence of TNC.</p
Identification of growth factors with an affinity towards TNCIII1â5.
<p>Binding of TNCIII1â5 to growth factors of different families, measured using indirect ELISA. Binding was measured in the presence of 4 ”M heparin (grey) and in its absence (black). Various growth factor families were tested, PDGF/VEGF family (A), FGF family (B), TGF-ÎČ family (C), IGF family and IGF-BPs (D), and others (E) (nâ=â3, mean±SD).</p
The effect of heparin concentration on the ability of TNCIII1â5 to bind selected growth factors.
<p>Using ELISA, the binding of TNCIII1â5 (10 nM) to various growth factors (A) PDGF-BB, (B) VEGF-A165, (C) FGF-2, and (D) BDNF was measured in the presence of increasing concentrations of heparin, 0.01 nM to 10,000 nM, with 100% representing binding of 10 nM TNCIII1â5 without the presence of heparin. The binding of the growth factors to TNCIII1â5 reached a maximum at an intermediate heparin concentration for all growth factors tested (nâ=â3, mean±SD).</p
Determination of the specific growth factor-binding domain.
<p>The binding affinity of different (fusion) domains (GST-TNCIII3, GST-TNCIII4, GST-TNCIII5, GST-TNCIII3-4, GST-TNCIII4-5, GST-TNCIII3-5) to PDBF-BB was determined and compared to controls, GST only and BSA, using an indirect ELISA method. The TNC domain responsible for growth factor binding was mainly identified as domain TNCIII5, with TNCIII4 contributing to a lesser extent. (nâ=â3, mean±SD).</p
Equilibrium binding constants (K<sub>D</sub>), and association (k<sub>on</sub>) and dissociation (k<sub>off</sub>) rates were determined using surface plasmon resonance (SPR) and fitting the data to Langmuir binding kinetics for binding of immobilized TNCIII1â5 to TGF-ÎČ1 and NT-3.
*<p>Not applicable (NA) because the data was not interpretable using standard Langmuir binding kinetics for FGF-2 and PDGF-BB.</p
Production and characterization of TNCIII1-5.
<p>(A) Top: schematic of structural domains of full length human tenascin C (TNC), with major integrin binding and heparin binding domains. Bottom: schematic of the designed TNCIII1-5 fragment. (B) SDS-PAGE gel of purified TNCIII1â5 with the molecular weight of 53 kD, and the different GST-TNCIII (fusion) domains (GST-TNCIII3, GST-TNCIII4, GST-TNCIII5, GST-TNCIII3-4, GST-TNCIII4-5, GST-TNCIII3-5). (C) Amino acid sequence of TNCIII1â5. (D) Graphical depiction of isoelectric points of the TNCIII domains.</p
Reduction-Sensitive Tioguanine Prodrug Micelles
Colloidal drug and prodrug conjugates have unique targeting
characteristics
for tumor vasculature from the blood and for the lymphatics draining
a tissue injection site. Tioguanine and tioguanine-generating prodrugs
have been investigated as anticancer and immunosuppressive agents,
including use in cancer immunotherapy. Recently we developed block
copolymers of polyÂ(ethylene glycol)-<i>bl</i>-polyÂ(propylene
sulfide) that self-assemble in aqueous solutions to form micellar
structures. Since the polymers carry a free terminal thiol group resulting
from the ring-opening polymerization of the propylene sulfide monomer,
we sought to prepare prodrug block copolymers with tioguanine linked
by a reduction-sensitive disulfide bond. The synthesis involved a
disulfide exchange between the oxidized form of tioguanine and the
polymer. Spectroscopic data is presented to support the proposed reaction.
The polymers self-assembled when dispersed in water to form tioguanine
prodrug micelles with a size range between 18 and 40 nm that released
tioguanine in response to cysteine and serum as shown spectroscopically.
In comparison with a polyÂ(ethylene glycol) prodrug polymer, we show
that the rate of tioguanine release can be controlled by changing
the polyÂ(propylene sulfide) block length and that the tioguanine remains
bioactive with cultured cells
Preparation of Well-Defined Ibuprofen Prodrug Micelles by RAFT Polymerization
Nonsteroidal
anti-inflammatory drugs (NSAIDs) are widely used to
treat acute pain, fever, and inflammation and are being explored in
a new indication in cancer. Side effects associated with long-term
use of NSAIDs such as gastrointestinal damage and elevated risk of
stroke, however, can limit their use and exploration in new indications.
Here we report a facile method to prepare well-defined amphiphilic
diblock copolymer NSAID prodrugs by direct reversible additionâfragmentation
transfer (RAFT) polymerization of the acrylamide derivative of ibuprofen
(IBU), a widely used NSAID. The synthesis and self-assembling behavior
of amphiphilic diblock copolymers (PEG-PIBU) having a hydrophilic
polyÂ(ethylene glycol) block and a hydrophobic IBU-bearing prodrug
block were investigated. Release profiles of IBU from the micelles
by hydrolysis were evaluated. Furthermore, the antiproliferative action
of the IBU-containing micelles in human cervical carcinoma (HeLa)
and murine melanoma (B16âF10) cells was assessed
Human clonal urothelial cells arising from single ureteral cell.
<p>(A) Schematics showing the passage from the single selected cell to the in vivo implantated clonal cell pellet. (B,C and D) Human urothelial holoclone, meroclone and paraclone cultures arising from one single ureteral cell. (E and F) Growth curve of a human urothelial holoclone (G) <i>In vivo</i> urothelial differentiation of human ureteral urothelial holoclone pellets implanted into the subcapsular space of the Swiss nu/nu mice, expressing cytokeratin 7, uroplakin-2 and uroplakin 3 (scale bars, 10 ”m). Note the âmicro-bladderâ like structure.</p
Porcine clonal urothelial cells arising from a single urethral cell.
<p>(A, B and C) Porcine urethelial holoclone, meroclone and paraclone cultures arising from a single urethral cell. (D and E) Growth curves of a porcine urothelial holoclone. (F, G) <i>In vivo</i> urothelial differentiation of porcine urethral urothelial holoclonal cell pellets implanted into the subcapsular space of the Swiss nu/nu mice, expressing cytokeratin 7, uroplakin-2 and uroplakin 3 (scale bars, 10 ”m). Note the âmicro-bladderâ like structure.</p