Influence of gelatin complexation on cell proliferation activity and proteolytic resistance of basic fibroblast growth factor

The objective of this study is to investigate the influence of gelatin complexation on the biological activity of basic fibroblast growth factor (bFGF) and its resistance to trypsin digestion. When bFGF was mixed at 37°C with acidic gelatin with an isoelectric point(IEP)of 5.0, the activity to promote in vitro proliferation of BHK cells became lower compared with that of free bFGF,in contrast to mixing with the basic gelatin with an IEP of 9.0. A maximum reduction in the bFGF activity was observed for the bFGF-gelatin complex prepared at a mixing molar ratio of 1/1. The bFGF activity of cell proliferation reduced at the initial period after mixing with the acidic gelatin at 37°C, followed by no substantial change. Complexation with the acidic gelatin at 4°C had no influence on the bFGF activity, irrespective of the bFGF/gelatin ratio and complexation time. The biological activity of bFGF was reduced by the trypsin treatment, but the reduced extent was suppressed through gelatin complexation at 37°C. In an electrophoresis study, the protective effect of gelatin complexation on the trypsin digestion was also confirmed in terms of the molecular weight loss. It is possible that the complexing gelatin covers bFGF molecules, resulting in suppression of their interaction with the cell surface receptor as well as protection from their enzymatic attack.     

Vascularization effect of basic fibroblast growth factor released from gelatin hydrogels with different biodegradabilities.

Biodegradable gelatin hydrogels were prepared through the glutaraldehyde crosslinking of acidic gelatin with an isoelectric point (IEP) of 5.0 and the basic gelatin with an IEP of 9.0. The hydrogel water content was changed by the concentration of both gelatin and glutaraldehyde, used for hydrogel preparation. An aqueous solution of basic fibroblast growth factor (bFGF) was sorbed into the gelatin hydrogel freeze-dried to obtain a bFGF-incorporating gelatin hydrogel. Irrespective of the hydrogel water content, approximately 30% of the incorporated bFGF was released from the bFGF-incorporating acidic gelatin hydrogel, within the first day into phosphate-buffered saline solution at 37 degrees C, followed by no substantial release. Probably, the basic bFGF complexed with the acidic gelatin through poly-ion complexation would not be released under the in vitro non-degradation condition of gelatin. On the contrary, almost 100% of the incorporated bFGF was initially released from all types of basic gelatin hydrogels. This is due to the simple diffusion of bFGF because of no complexation between bFGF and the basic gelatin. When implanted subcutaneously into the mouse back, bFGF-incorporating acidic and basic gelatin hydrogels with higher water contents were degraded with time faster than those with lower water contents. Significant neovascularization was induced around the implanted site of the bFGF-incorporating acidic gelatin hydrogel. The induction period prolonged with the decrease in hydrogel water content. On the other hand, such a prolonged vascularization effect was not achieved by the bFGF-incorporating basic gelatin hydrogel and the hydrogel initially exhibited less enhanced effect, irrespective of the water content. These findings indicate that the controlled release of biologically active bFGF is caused by biodegradation of the acidic gelatin hydrogel, resulting in induction of vascularization effect dependent on the water content. It is possible that only the transient vascularization by the basic gelatin hydrogel is due to the initial large burst in bFGF release, probably because of the down regulation of bFGF receptor.     

Neovascularization effect of biodegradable gelatin microspheres incorporating basic fibroblast growth factor

Biodegradable microspheres were prepared through glutaraldehyde cross-linking of gelatin without using any surfactants as a carrier matrix of basic fibroblast growth factor (bFGF). In the in vitro system, bFGF was sorbed to microspheres of acidic gelatin with an isoelectric point (IEP) of 5.0, but not to those of basic gelatin with an IEP of 9.0. The rate of bFGF sorption to the acidic gelatin microsphere in phosphate-buffered saline solution (pH 7.4) was smaller than that in water. Following incorporation of bFGF into the microspheres at 4 degrees C for 12 h, bFGF release from the bFGF-incorporating microspheres was studied. Approximately 30% of incorporated bFGF was released from the acidic gelatin microsphere within the initial 3 h, followed by no substantial release, whereas the basic gelatin microsphere released almost completely the incorporated bFGF within 1 day. It is likely that when basic bFGF molecules were immobilized to the acidic gelatin constituting microspheres through polyion complexation, they were not readily released under the in vitro nondegradation condition of gelatin. Incorporation of anionic carboxylmethyl cellulose (CMC) into the acidic gelatin microspheres reduced the amount of bFGF desorbed initially. This indicates that the initial burst is ascribed to free bFGF which is not ionically interacted with the acidic gelatin. CMC will function as a bFGF sorbent to suppress the initial leakage from the microspheres. When injected subcutaneously into the mouse back, bFGF-incorporating acidic gelatin microspheres were degraded over time and induced neovascularization around the injection site, in marked contrast to bFGF in the solution form. CMC incorporation slowed down the biodegradation and vascularization effect of bFGF-incorporating gelatin microspheres. It was concluded that the gelatin microsphere was a promising carrier matrix of bFGF to enhance the vascularization effect.     

Interaction of hepatocyte growth factor with gelatin as the carrier material

The objective of this study was to physicochemically investigate the interaction between hepatocyte growth factor (HGF) and acidic gelatin compared with that between HGF and basic gelatin or heparin. Gelatin- or heparin-immobilized agarose beads were prepared and HGF interaction with them was evaluated by Scatchard binding assay. The dissociation constant of HGF with the acidic gelatin was about 2-3 orders of magnitude higher than that of heparin. The cell proliferation assay revealed that the proliferation promotion activity of HGF complexed with the acidic gelatin was detected, although it was lower than that of original HGF. The ability of HGF to enhance the cell proliferation was reduced by the trypsin treatment, although the extent of the reduction was significantly suppressed by HGF complexation with acidic gelatin. Electrophoresis experimentally confirmed enhanced resistance to the molecular mass loss of HGF by gelatin complexation. Moreover, the recognized level of an antibody to HGF was reduced by the complexation with the acidic gelatin, indicating that the acidic gelatin is present around HGF molecules. It is possible that the HGF molecule is covered with the acidic gelatin, resulting in protection from enzymatic digestion.     

Interaction of hepatocyte growth factor with gelatin as the carrier material

The objective of this study was to physicochemically investigate the interaction between hepatocyte growth factor (HGF) and acidic gelatin compared with that between HGF and basic gelatin or heparin. Gelatin- or heparin-immobilized agarose beads were prepared and HGF interaction with them was evaluated by Scatchard binding assay. The dissociation constant of HGF with the acidic gelatin was about 2–3 orders of magnitude higher than that of heparin. The cell proliferation assay revealed that the proliferation promotion activity of HGF complexed with the acidic gelatin was detected, although it was lower than that of original HGF. The ability of HGF to enhance the cell proliferation was reduced by the trypsin treatment, although the extent of the reduction was significantly suppressed by HGF complexation with acidic gelatin. Electrophoresis experimentally confirmed enhanced resistance to the molecular mass loss of HGF by gelatin complexation. Moreover, the recognized level of an antibody to HGF was reduced by the complexation with the acidic gelatin, indicating that the acidic gelatin is present around HGF molecules. It is possible that the HGF molecule is covered with the acidic gelatin, resulting in protection from enzymatic digestion.     

Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor.

Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a confluent monolayer of ECs. Human umbilical vein endothelial cells (HUVECs) were grown on bFGF-loaded albumin-heparin conjugate bound to CO2 gas-plasma-treated polystyrene. In the order of 2-3 ng/cm2 bFGF had to be immobilized to form a confluent monolayer of HUVECs. The most prominent effect of surface-immobilized bFGF was stimulation of the proliferation shortly after seeding, resulting within 3 days in confluent cell monolayers with high density. In contrast, in cultures with 0.3 ng/mL bFGF in the medium instead of bFGF bound to the surface, it took almost a week before the cell layers reached confluency. Binding of bFGF to heparin and the biological activity of bFGF towards ECs were not influenced by the (radio-)labeling of bFGF with iodine. However, only a minor part of the bFGF used in this study displayed heparin affinity. Furthermore, degradation and multimerization of labeled bFGF in time occurred when the growth factor was stored at 20 degrees -37 degrees C. This limits the use of labeled bFGF to short-term (hours) experiments. In conclusion, bFGF loading of vascular graft surfaces through complexation of bFGF with a heparin-containing matrix probably will lead to more rapid formation of a confluent monolayer of ECs on graft surfaces upon seeding of the cells.     

Controlled release of growth factors based on biodegradation of gelatin hydrogel

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-beta1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-beta1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.     

Controlled release of growth factors based on biodegradation of gelatin hydrogel

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-β1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-β1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.     

Affinity evaluation of gelatin for hepatocyte growth factor of different types to design the release carrier

The objective of this study was to investigate the physicochemical interaction of hepatocyte growth factor (HGF) and its variant with 5 amino-acid residues deleted (dHGF) with an acidic gelatin for the design of factors release from the gelatin hydrogel. When the interaction of HGF or dHGF with gelatin-immobilized agarose beads was evaluated by Scatchard binding assay, the dissociation constant of dHGF was higher than that of HGF, although the two proteins had a similar binding ratio. dHGF was released more rapidly from the hydrogel of acidic gelatin than HGF. In vivo release study with ¹²⁵I-labeled HGF or dHGF in mice subcutis showed that HGF was released from the gelatin hydrogel as a result of hydrogel degradation. In contrast, dHGF was rapidly released by a simple diffusion from the gelatin hydrogel. From electrophoresis experiments, mixing with the acidic gelatin enabled HGF to complex and suppressing the trypsin-digested molecular weight loss, in marked contrast to that of dHGF. In addition, the percentage of HGF recognized by the antibody was reduced by the gelatin complexation, but that of dHGF was not. We conclude that unlike dHGF, HGF has a strong affinity for the acidic gelatin, resulting in the controlled release of HGF accompanied with hydrogel degradation of the release carrier.     

Affinity evaluation of gelatin for hepatocyte growth factor of different types to design the release carrier

The objective of this study was to investigate the physicochemical interaction of hepatocyte growth factor (HGF) and its variant with 5 amino-acid residues deleted (dHGF) with an acidic gelatin for the design of factors release from the gelatin hydrogel. When the interaction of HGF or dHGF with gelatin-immobilized agarose beads was evaluated by Scatchard binding assay, the dissociation constant of dHGF was higher than that of HGF, although the two proteins had a similar binding ratio. dHGF was released more rapidly from the hydrogel of acidic gelatin than HGF. In vivo release study with 125I-labeled HGF or dHGF in mice subcutis showed that HGF was released from the gelatin hydrogel as a result of hydrogel degradation. In contrast, dHGF was rapidly released by a simple diffusion from the gelatin hydrogel. From electrophoresis experiments, mixing with the acidic gelatin enabled HGF to complex and suppressing the trypsin-digested molecular weight loss, in marked contrast to that of dHGF. In addition, the percentage of HGF recognized by the antibody was reduced by the gelatin complexation, but that of dHGF was not. We conclude that unlike dHGF, HGF has a strong affinity for the acidic gelatin, resulting in the controlled release of HGF accompanied with hydrogel degradation of the release carrier.     

Effects of bFGF incorporated into a gelatin sheet on wound healing

Basic fibroblast growth factor (bFGF) is well known to promote the proliferation of almost all cells associated with wound healing. However, as the activation duration of bFGF is very short in vivo, we incorporated bFGF into an acidic gelatin hydrogel and studied the sustained release of bFGF in vivo. In addition, we investigated the effects of the acidic gelatin sheet containing bFGF on wound healing. To distinguish wound contraction from neoepithelialization, we measured both the wound area and neoepithelium length. Other histological parameters such as thickness of granulation tissue and number of capillaries were also determined as indices of wound healing. Fibrous tissue was assessed using an Elastica van Gieson and Azan stain. A skin defect (1.5 x 1.5 cm) of full thickness was created on the back of each test mouse and the wound was covered with an acidic gelatin hydrogel, referred to as a gelatin sheet in this study (2 x 2 cm), with bFGF (100 microg/site) (A) or without bFGF (B). 1, 2, 3, 5, 7 and 14 days after covering, mice were killed and an enzyme-linked immunosorbent assay (ELISA) was performed to estimate the concentration of bFGF in the plasma. In another experiment, each wound was covered with (A), (B) or a hydrogel dressing (control group, C) and the wound area was measured 1 or 2 weeks postoperatively with a computer planimeter. The histological parameters, as mentioned above, were assessed using a light microscope. Sustained release of bFGF from the gelatin sheet was observed and the gelatin sheet containing bFGF promoted neoepithelialization, granulation, neovascularization and wound closure. This gelatin sheet containing bFGF was concluded to be effective for wound healing and promising for clinical use.     

Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis

The objective of this study was to evaluate the effect of incorporation of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres into an artificial dermis on the regeneration of dermis-like tissues. When used in the free form in vivo, bFGF cannot induce sufficient wound healing activity, because of its short half-life. Therefore, sustained release of bFGF was achieved by impregnation into biodegradable gelatin microspheres. A radioisotope study revealed that incorporation of bFGF-impregnated gelatin microspheres significantly prolonged in vivo retention of bFGF in the artificial dermis. Artificial dermis with incorporated bFGF-impregnated gelatin microspheres or bFGF in solution was implanted into full-thickness skin defects on the back of guinea pigs (1.5 cm x 1.5 cm) (n = 4). Incorporation of bFGF into the artificial dermis accelerated fibroblast proliferation and capillary formation in a dose-dependent manner. However, the accelerated effects were more significant with the incorporation of bFGF-impregnated gelatin microspheres than with free bFGF at doses of 50 microg or higher. We conclude that the gelatin microsphere is a promising tool to accelerate bFGF-induced tissue regeneration in artificial dermis.     

Basic fibroblast growth factor in ovulatory cycle and postmenopausal human endometrium

Biopsies of human endometrium were studied for the presence of basic fibroblast growth factor (bFGF). An immunoreactive Mr 18,000 bFGF-like molecule was detected at high levels both in ovulatory cycle and postmenopausal endometrium. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, its capacity to induce plasminogen activator production and cell proliferation in endothelial GM 7373 cells, and its cross-reactivity with various anti-bFGF antibodies. The levels of endometrial bFGF do not change during the menstrual cycle but they increase significantly after menopause, as evaluated both by biological and immunological assays. Lower levels of an acidic FGF-like activity were also evident in ovulatory cycle endometrium but, at variance with bFGF, no significant increase of this activity was observed in postmenopausal endometrium. These data represent the first characterization of a polypeptide growth factor present in human endometrium.     

Biodegradation of hydrogel carrier incorporating fibroblast growth factor.

In vivo release of basic fibroblast growth factor (bFGF) from a biodegradable gelatin hydrogel carrier was compared with the in vivo degradation of hydrogel. When gelatin hydrogels incorporating 125I-labeled bFGF were implanted into the back subcutis of mice, the bFGF radioactivity remaining decreased with time and the retention period was prolonged with a decrease in the water content of the hydrogels. The lower the water content of 125I-labeled gelatin hydrogels, the faster both the weight of the hydrogels and the gelatin radioactivity remaining decreased with time. The decrement profile of bFGF remaining in hydrogels was correlated with that of hydrogel weight and gelatin radioactivity, irrespective of the water content. Subcutaneous implantation of bFGF-incorporating gelatin hydrogels into the mice induced significant neovascularization. The retention period of neovascularization became longer as the water content of the hydrogels decreased. To study the decrease of activity of bFGF when implanted, bFGF-incorporating hydrogels were placed in diffusion chamber and implanted in the mouse subcutis for certain periods of time. When hydrogels explanted from the mice were again implanted, significant neovascularization was still observed, indicating that most of the biological activity of bFGF was retained in the hydrogels. It was concluded that, in our hydrogel system, biologically active bFGF was released as a result of in vivo degradation of the hydrogel. The release profile was controllable by changing the water content of hydrogels.     

Effect of culture substrates and fibroblast growth factor addition on the proliferation and differentiation of rat bone marrow stromal cells

This study is an investigation of the proliferation and differentiation of bone marrow stromal cells (BMSCs) on film substrates with different surface properties. Films of noncharged polymers with several water wettabilities; cell culture plates coated with collagen type I or IV, gelatin, or basic fibroblast growth factor (bFGF); and glass were used. BMSCs isolated from rat bone marrow were cultured on the various substrates in medium with dexamethasone [Dex(+)] or without dexamethasone [Dex(-)] to assess cell proliferation and differentiation. The number of proliferated BMSCs depended on the water wettability of substrates, although the cell number was greater in Dex(-) medium than in Dex(+) medium. Protein-coated substrates exhibited a high proliferation rate compared with noncoated substrates. Alkaline phosphatase (ALP) activity increased with increasing cell number, whereas ALP activity per cell correlated well with cell number. When cultured in Dex(+) medium containing bFGF or on culture plates coated with bFGF, BMSC proliferation tended toward enhancement with an increase in the amount of bFGF added in solution form, whereas it did not depend on the amount of bFGF in coated form. On the other hand, ALP activity and calcium content of BMSCs became maximal with bFGF coated at about 1 x 10(3) to 2 x 10(3) ng, in contrast to bFGF in solution form. Irrespective of the amount of bFGF, ALP activity and calcium content levels for bFGF in coated form were higher than for bFGF in solution form. It is concluded that the type of culture substrate and the manner of addition of bFGF affect the proliferation and differentiation of BMSCs.     

Effect of FGFs on adult bovine Muller cells: proliferation, binding and internalization.

A new method for culturing retinal Muller cells from adult bovine tissue is described. The identification of these glial cells was based on immunocytochemical analysis of specific Muller cell markers. Cultured cells from fourth to ninth passage showed positive labelling for S 100 protein, carbonic anydrase (CAA), glutamine synthetase (GS), alpha cristallin (alpha C) and polyclonal glial fibrillary acidic protein (GFAP) antibody, but were negative for both monoclonal GFAP antibody and also for Muller cells in the retina. Investigation of the effect of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF) on the proliferation of the Muller cells revealed that bFGF was the most potent mitogen (EC50 = 14 pM). Binding data revealed the presence of two classes of binding sites for aFGF and bFGF: (1) a high affinity binding site (Kd of 14 pM and 27 pM for aFGF and bFGF respectively); (2) a low affinity binding site (Kd of 3.2 nM and 0.6 nM for aFGF and bFGF respectively with great variability in the number of binding sites). In addition, the cross-linking experiments revealed the presence of high molecular weight FGF receptors (110-140 kDa). After aFGF or bFGF binding to Muller cells, aFGF and bFGF-cell surface receptors were rapidly downregulated with a half-life for disappearance of 35-50 min. Internalization and degradation of 125I-bFGF bound to the Muller cell receptors did not occur at 4 degrees C. At 37 degrees C, however, there was a rapid decrease in receptor-bound 125I-bFGF due to the downregulation of bFGF receptors. Concomitantly 125I-bFGF appeared inside the Muller cells. After 2 h, 125I-bFGF began to be degraded and after 6 h three fragments of 16 kDa, 8 kDa and 5.5 kDa were discernible. Degradation of bFGF appeared to occur in the lysosomal compartment since it was inhibited by chloroquine, an inhibitor of lysosomal proteases; aFGF internalization and degradation followed the same kinetics as bFGF with the appearance of 7 kDa and 5 kDa fragments. These results suggest that Muller cells may be the target for aFGF and bFGF contained in other cells of the retina. The fact that aFGF could be released from rod outer segment by a phosphorylation-dependent mechanism, and that apical prolongation of the Muller cells is connected with the photoreceptor cells suggest that these factors may be the mediators involved in the communication between glial cells and neurons.     

Transglutaminase crosslinked gelatin as a tissue engineering scaffold

Gelatin is one of the most commonly used biomaterials for creating cellular scaffolds due to its innocuous nature. In order to create stable gelatin hydrogels at physiological temperatures (37 degrees C), chemical crosslinking agents such as glutaraldehyde are typically used. To circumvent potential problems with residual amounts of these crosslinkers in vivo and create scaffolds that are both physiologically robust and biocompatible, a microbial transglutaminase (mTG) was used in this study to enzymatically crosslink gelatin solutions. HEK293 cells encapsulated in mTG-crosslinked gelatin proliferated at a rate of 0.03 day(-1). When released via proteolytic degradation with trypsin, the cells were able to recolonize tissue culture flasks, suggesting that cells for therapeutic purposes could be delivered in vivo using an mTG-crosslinked gelatin construct. Upon submersion in a saline solution at 37 degrees C, the mTG-crosslinked gelatin exhibited no mass loss, within experimental error, indicating that the material is thermally stable. The proteolytic degradation rate of mTG-crosslinked gelatin at RT was slightly faster than that of thermally-cooled (physically-crosslinked) gelatin. Thermally-cooled gelatin that was subsequently crosslinked with mTG resulted in hydrogels that were more resistant to proteolysis. Degradation rates were found to be tunable with gelatin content, an attribute that may be useful for either long-time cell encapsulation or time-released regenerative cell delivery. Further investigation showed that proteolytic degradation was controlled by surface erosion.     

Thermoresponsive artificial extracellular matrix: N-isopropylacrylamide-graft-copolymerized gelatin

Poly(N-isopropylacrylamide)-graft-copolymerized gelatin (PNIPAM-gelatin) was prepared by iniferter-based photopolymerization of multiply derivatized dithiocarbamylated gelatin. PNIPAM-gelatins exhibited low critical solution temperature (LCST) immediately below the physiological temperature. PNIPAAm-gelatin-coated dishes induced cell adhesion at 37 degrees C but incomplete detachment at room temperature, whereas dishes coated with PNIPAM or a mixture of PNIPAAm and gelatin showed little cell adhesion. The mixture of PNIPAAm-gelatin and PNIPAAm induced cell adhesion at 37 degrees C and detachment at 20 degrees C: the degrees of cell adhesion and detachment depended on the mixed ratio of PNIPAAm-gelatin and PNIPAAm. Complete thermoresponsive adhesion and detachment were found for the mixture containing a small fraction of PNIPAAm-gelatin (approximately 5 wt% with respect to PNIPAAm; gelatin content in the mixture is 2.7 wt%). Such a mixture may serve as thermoresponsive cell matrix for fabrication of a tissue-engineered device.     

Activation of botulinum C2 toxin by trypsin.

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two dissimilar protein components, designated components I and II. The biological activity of C2T is enhanced by treating the toxin with trypsin. This activation of C2T is observed as a result of mixing untrypsinized component I and trypsinized component II but not as a result of mixing trypsinized component I and untrypsinized component II. The data presented here show that the maximum lethality of C2T, determined by mixing untrypsinized component I and trypsinized component II, was attained by treating component II with trypsin at a ratio of 10:1 on a protein basis for 30 min at 35 degrees C at pH 7.5. The activation of component II was always accompanied by a change in the molecular weight of the component from 101,000 to 88,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the gel filtration of trypsinized component II resulted in the separation of two active components, with apparent molecular weights, estimated from the elution volume by gel filtration, of 365,000 and 74,000. The high-molecular-weight component II had hemagglutination and hemolytic activities, whereas the low-molecular-weight component II has only hemagglutination activity. These two molecular species of active component II had approximately the same lethality, when mixed with component I, and gave a single band in SDS-PAGE, with a molecular weight of 88,000, the same as that of trypsin-activated component II under different reaction conditions. The results indicate that the activation of C2T by trypsin is due to the molecular conversion of component II from molecular weight 101,000 to 88,000 as determined by SDS-PAGE and that the trypsin-activated component II tends to form an oligomer of the active component II.     

Purification and characterization of proteases secreted by transforming schistosomula of Schistosoma mansoni

Schistosomula of Schistosoma mansoni which are mechanically transformed at 4 degrees C and are then incubated at 37 degrees C in defined medium spontaneously secrete two proteases, a major one of 28 kDa and a minor one of 60 kDa. These were purified by ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel AcA 54 with yields of 33% and 29%, respectively. Both appeared as single bands by silver staining following sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. The 28 kDa protease is a glycoprotein that has a pI of 11 or higher and an optimal activity around pH 9.0. It cleaves casein, gelatin and human C3 and C3b. It is metal-ion independent and is inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, soy-bean trypsin inhibitor, alpha 1 antitrypsin, Zn2+ ions, sodium dodecyl sulphate and normal human serum. The 60 kDa protease is a glycoprotein with a pI of 9.2. It can also cleave casein and gelatin and its activity is inhibited by phenylmethanesulfonyl fluoride but not by diisopropyl fluorophosphate or sodium dodecyl sulphate. We suggest that these proteases may play a role during cercarial penetration of the skin and in shedding of the cercarial glycocalyx.