Linda L. Dart

Transforming growth factor β levels in rat wound chambers

Exogenous TGF-β accelerates healing in both normal and doxorubicin-treated rats, but whether it plays an intrinsic role in the natural healing process is unknown. Subcutaneous wound chambers in 16 F344 rats were aspirated from postwounding Day 3 through Day 16 for TGF-β levels and cytology. A soft agar assay and a competitive radioreceptor binding assay were used to determine TGF-β levels. Papanicolau staining and differential cell counts were used to determine cytology. Results were similar using either method for the determination of TGF-β levels. With the sensitive radioreceptor assay, low TGF-s levels on postwounding Day 4, mean 2.6 ng/ml, rose to a peak mean level of 20.4 ng/ml on Day 7 and fell significantly from peak level to a level of 5.4 ng/ml of Day 16. All TGF-β levels for postwounding Days 6 through 14 were significantly increased over the baseline TGF-β levels of Days 4 and 5 (P < 0.05). Day 16 TGF-β levels were not different from baseline. Cytologic changes were characterized by a linear decrease in total neutrophil count over the exam period and a concurrent linear increase in total lymphocyte and macrophage counts. TGF-β levels changed in a bell-shaped temporal sequence during healing, apparently unrelated to percentage lymphocyte, macrophage, or neutrophil count. Peak TGF-β levels occurred during the fibroblast proliferation and collagen synthesis phase of healing. This study presents the first evidence that TGF-β is present in a healing wound and suggests that it may be an intrinsic mediator of the healing process.

Sandwich Enzyme-Linked Immunosorbent Assays (Selisas) Quantitate and Distinguish Two Forms of Transforming Growth Factor-Beta (TGF-β1 and TGF-β2) in Complex Biological Fluids

We have developed sandwich enzyme-linked immunosorbent assays (SELISAs) for TGF-beta 1 and TGF-beta 2 using both turkey and rabbit neutralizing polyclonal antibodies against native TGF-beta s. Each assay is based on the binding of two different antibodies to distinct epitopes of a single TGF-beta molecule. With these assays, TGF-beta types 1 and 2 can each be specifically quantitated in complex biological fluids, with detection limits of 2-5 pg. TGF-beta 3 and TGF-beta 5 either do not cross-react or cross-react very poorly in these assays. TGF-beta 1.2 heterodimer, although 50-80% neutralized by either TGF-beta 1 or TGF-beta 2 antibodies, shows only a 1.5 and 3.7% cross-reactivity in the TGF-beta 1 and TGF-beta 2 SELISAs, respectively. The SELISAs reported here represent the most specific, rapid, and precise assays for TGF-beta 1 and TGF-beta 2 reported thus far.

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

Abstract The metabolism of 13-cis-[11-3H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.

Metabolism of all-trans-retinyl acetate to retinoic acid in hamster tracheal organ culture

All-trans-[3H]retinyl acetate has been shown to be metabolized to all-trans-[3H]retinoic acid in a target tissue of vitamin A action, the hamster trachea in organ culture. That the compound produced is indeed all-trans-retinoic acid is demonstrated by chromatography of the biosynthetically produced retinoic acid with synthetic all-trans-retinoic acid in two different high-pressure liquid chromatographic systems, either as the free acids in a reverse-phase system or as the methyl esters in a normal-phase system. The all-trans-[3H]retinoic acid was also found in the tracheal epithelium and cartilage as well as in the medium. In addition the tracheal tissue also contained retinyl palmitate and other esters. Finally, further in vitro metabolism of [3H]retinyl acetate paralleled the metabolism of [14C]retinoic acid suggesting that these two compounds are being metabolized through similar pathways.

Evidence for Differential Regulation of TGFβ1 and TGFβ2 Expression in Vivo by Sandwich Enzyme‐linked Immunosorbent Assays

We have developed sandwich enzyme-linked immunosorbent assays (SELISAs) for TGFPl and TGFp2 using both turkey and rabbit neutralizing polyclonal antibodies against native TGFps. Each assay is based on the binding of two different antibodies to distinct epitopes of a single TGFD molecule. For these assays, Nunc Maxisorb (Nunc, Denmark) microtiter plates are first coated with either affinity-purified anti-TGFo1 or anti-TGFP2 rabbit antibodies, and the TGFps are then added and allowed to be anchored to wells by the primary antibodies. The exposed epitopes of the immobilized TCFp1 and TGFP2 serve as binding sites for secondary affinity-purified anti-TGFpl and anti-TGFp2 antibodies, respectively. The bound secondary antibody is then detected by phosphatase activity following the binding of phosphatase-linked anti-turkey IgG. With these assays, TGFP types 1 and 2 can each be specifically quantitated in complex biological fluids with detection limits of 2 to 5 pg. Conditioned medium can be assayed after acid activation, and tissue and serum can be assayed after extraction with acid-ethanol. Various steroid hormones and peptide growth modulators do not interfere in these SELISAs. Because each of these SELISAs are based on the recognition of two independent epitopes, they exhibit much greater ligand specificity relative to immunoassays that are based on the recognition of only a single epitope. Such enhanced specificity is clearly demonstrated by our observation that only 1.5 and 3.7% of TCFpl.2 heterodimer is scored in the TGFpl and TGF(32 SELISAs, respectively, in contrast to 82 and 50% neutralization of biological activity by anti-TCFpl and antiTGFp2, respectively. Chicken TGFP3 and frog TCFp5 do not crossreact in these SELISAs, further supporting the high specificity of these SELISAs. Therefore, the many favorable features of these SELISAs make them the method of choice for rapid, accurate, and precise measurement of mFD1 and E F P 2 in complex biological fluids. The levels of E F p l and TGFp2 in sera from various animals were measured by these SELISAs (TABLE 1). Our results from this study demonstrate that TGFpl is the

Inhibition of transforming growth factor-induced cell growth in soft agar by oxidized polyamines

An inhibitor of the transforming growth factor-induced growth in soft agar of normally anchorage-dependent rat kidney fibroblasts has been detected in the acid-ethanol extracts of human placenta, bovine lung and kidney, and human rhabdomyosarcoma cells (A673). The inhibitor has been purified from human placenta by gel-filtration and cation-exchange chromatography followed by acetylation and HPLC. Acetylation destroys inhibitory activity and deacetylation, by treatment with 6 N HCl at 110 degrees C for 16 h, restores full activity. The purified compound has been identified as spermine by mass spectral and NMR analyses and by cochromatography on HPLC of the acetylated material with acetylated spermine. Both the compound isolated from the placenta and spermine and spermidine show approximately equal activity in inhibiting the transforming growth factor-induced growth of cells in soft agar with an ED50 of 0.7-1.1 microM, while putrescine displays no inhibitory activity. Evidence suggests that the polyamines must first be oxidized by serum polyamine oxidase before inhibition will occur. Acrolein, a product of polyamine oxidation, will also inhibit cell growth in soft agar with an ED50 of 6.8 microM. It is concluded that an oxidation product of spermine is responsible for the previously reported inhibition of colony growth in soft agar following treatment of normal fibroblasts with transforming growth factors.

Variables in the high-pressure cation-exchange chromatography of proteins☆

The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and p I range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.