"Matrigenin" activity from bovine bone--III. Effects on glycosaminoglycans and proteoglycans of human synovial cells in culture

1. Human synovial fibroblastic cells were cultured in the presence and absence of an extract from bovine bone containing "matrigenin" activity. The rate of incorporation of radioactivity into the glycosaminoglycans of the medium of "matrigenin"-treated cultures increased after 24 hr of incubation, compared to "controls". 2. Higher serum concentrations had a greater effect on the incorporation of radioactivity into hyaluronic acid synthesized by "matrigenin"-treated cultures, than by "controls". 3. Incorporation of radioactive precursors into the proteoglycans isolated from the medium was greater in the "matrigenin"-treated cultures than in "controls". The synthesis of a large mol. wt proteoglycan was specifically stimulated.     

"Matrigenin" activity from bovine bone--II. Effects on the glycosaminoglycans of bovine articular cartilage in culture

1. Bovine articular cartilage slices were studied in long term culture by periodically pulse-labelling the cultures with radiolabelled precursors of glycosaminoglycans and isolating the glycosaminoglycans from cartilage. 2. Pretreatment of the cartilage slices with bacterial collagenase resulted in stimulation of the incorporation of radioactivity into the glycosaminoglycans. 3. The addition of a fraction from bovine bone, enriched in "matrigenin" activity, to cultures of cartilage pretreated with collagenase resulted in an additional increase in the stimulation of incorporation of radioactivity.     

Coincubation of bovine synovial or capsular tissue with cartilage generates a soluble "Aggrecanase" activity

The culture of bovine synovial or capsular tissue generated proteoglycan-degrading activity. When these tissues were incubated with living or dead bovine articular cartilage significantly more proteoglycan-degrading activity was revealed. The activity was present in a soluble form and required protein synthesis for its generation. The conditioned medium did not contain matrixin activity, although experiments with proteinase inhibitors suggested that the activity was due to a metalloproteinase. Western blotting of the aggrecan fragments suggested cleavage of aggrecan within the interglobular domain at the "aggrecanase" site, but not at the major matrixin site. N-terminal sequencing confirmed cleavage of aggrecan at a number of glutamyl bonds, including the aggrecanase site in the interglobular domain. We conclude that cultured synovial or capsular tissue produces soluble aggrecanase and an enzyme which releases aggrecanase from cartilage, possibly by cleavage of a chondrocyte membrane-bound form of aggrecanase.     

The effect of solubilized bone matrix fractions from different mammalian species on glycosaminoglycan synthesis by cultured fibroblasts

Human and bovine bone matrices were extracted with salt solutions of different composition and the extracts tested for stimulation of incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the hyaluronic acid and chondroitin sulfate of the cell pellet, the cell surface and the medium fractions of human synovial cells in culture. Stimulatory activity was extracted with a solution of 0.3 M EDTA in 2.5 M NaCl from bovine but not human bone. Subsequent extraction of the residues with 4 M guanidinium hydrochloride yielded activity from both matrices. A major stimulation of incorporation of radioactivity was observed in the cell surface fractions. Human synovial cells constitute a more sensitive assay system for the stimulatory activity than rabbit synovial cells.     

Endogenous "ouabain-like" activity in bovine plasma

An endogenous inhibitor of the sodium pump has been detected and concentrated 1000-fold from bovine plasma. The steps of purification included deproteinization and extraction with methanol, removal of lipids by coextractions with a lipophilic solvent, desalting and further concentration by adsorption on C18-SepPack cartridges and HPLC fractionation on a weak anionic exchange column. The material isolated displaces 3H-ouabain from brain synaptosomes, inhibits red cell membrane Na,K-ATPase without inhibiting Mg-ATPase or Ca,Mg-ATPase. Deproteinization of plasma by boiling may lead to appearance of non-specific inhibitors. The procedures developed should now permit isolation of sufficient amount of material for further purification and structural characterization.     

TGF-?1 Enhances the BMP-2-Induced Chondrogenesis of Bovine Synovial Explants and Arrests Downstream Differentiation at an Early Stage of Hypertrophy

Background

Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-ß1) were investigated.

Methodology/Principal Findings

Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-ß1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume.

Conclusions/Significance

TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the stimulation protocol for the optimal chondrogenic differentiation of synovial explants.     

Localization and cytophotometric analysis of cathepsin B activity in unfixed and undecalcified cryostat sections of whole rat knee joints

Cathepsin B activity is demonstrated histochemically with a post-coupling method using Z-Arg-Arg-4-methoxy-2-naphthylamide as substrate and Fast Blue BB as coupling reagent in unfixed and undecalcified cryostat sections of whole rat knee joints. Sections were attached to transparent tape to keep the integrity of the tissue intact, such attachment being essential for precise precipitation of the final reaction product at sites of enzyme activity. Also essential was inclusion of polyvinyl alcohol in the enzyme incubation medium. High cathepsin B activity was found in osteoclasts, chondrocytes, fibroblasts, synovial cells, and bone marrow cells in knee joints after induction of arthritis. The final reaction product was precipitated as fine cytoplasmic granules probably corresponding to lysosomes. The reaction was specific because addition to the incubation medium of selective inhibitors of cathepsin B-like activity completely blocked the activity. The amount of final reaction product in synovium and in bone marrow cells was analyzed cytophotometrically. Specific formation of final reaction product was linear with incubation time up to 60 min at 37 degrees C and with section thickness up to 12 microns. Variation of the substrate concentration in the incubation medium revealed a KM value of 1.86 +/- 0.36 mM in synovial cells and 2.48 +/- 0.51 mM in bone marrow cells and Vmax values (expressed as mean integrated absorbance) of 1.18 +/- 0.10 in synovial cells and 1.02 +/- 0.11 in bone marrow cells. Both KM and Vmax values were significantly different in synovial cells and bone marrow cells (p less than 0.01) which could be owing to the presence of different isoenzymes in these tissues. We conclude that the described post-coupling method is sufficient to yield precise localization and that the method is valid for quantitative purposes.     

Regulation of natural killer cell activity by macrophages in the rheumatoid joint and peripheral blood

Recently, in another study, we observed that indomethacin, a prostaglandin synthetase inhibitor, significantly increased NK activity in both normal and rheumatoid arthritis (RA) peripheral blood (PB) but not in RA synovial fluid (SF). Because macrophages are a major source of prostaglandins, we examined the effect of macrophage-enriched adherent cells (AC) on NK activity as measured by a 3-hr Cr-release assay with K 562 cells. The removal of AC resulted in increased (p less than 0.01) NK activity in both normal and RA PB. In contrast, the removal of AC from RA SF resulted in a significant decrease (p less than 0.001) of NK activity. By using only nonadherent cells (NAC), NK activity in RA SF and synovial tissue (ST) was significantly reduced when compared to autologous RA PB (p less than 0.001). Enhancement of NK activity of SF NAC by both poly I:C and IL 2 was not dependent on AC. Mixing experiments demonstrated that the addition of synovial AC for 16 hr increased NK activity of synovial NAC to a level similar to that of unseparated mononuclear cells, whereas autologous PB AC suppressed NK activity of PB NAC. PB AC, when added to SF NAC, also increased NK activity. Supernatants from synovial mononuclear cells were stimulatory of synovial NAC NK activity, whereas normal PB mononuclear supernatants were suppressive. These observations document 1) a significant reduction of NAC-mediated NK activity in the rheumatoid joint as compared to PB from the same patient, and 2) that AC modulate NK activity differently in the rheumatoid joint as compared to RA or normal PB.     

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

Introduction

Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients.

Methods

An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined.

Results

Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur.

Conclusions

We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

Purification of uricase by biospecific adsorption-desorption

A simple procedure for the purification of uricase from bovine kidney is described. The procedure involves the following steps: 1) processing of kidney mince by borate/butanol, 2) ammonium sulphate precipitation, and 3) biospecific adsorption-desorption. The adsorbents were prepared by chemical attachment of urate or xanthine to agarose gel beads. The desorption was performed by a xanthine solution. The adsorption-desorption procedure resulted in an 11 000-12 000-fold purification. The specific activity of the purified uricase was 19.8 U/mg using either "urate" adsorbent. The recovery was about 70%. The adsorbents were also used for the purification of commercial uricase preparations from hog liver. In this case the purified uricase also possessed a specific activity of 19.8 U/mg. The products were homogenous as judged by gradipore electrophoresis and gel filtration.     

Synovial Fluid Progenitors Expressing CD90+ from Normal but Not Osteoarthritic Joints Undergo Chondrogenic Differentiation without Micro-Mass Culture

Objective

Mesenchymal progenitor cells (MPCs) can differentiate into osteoblasts, adipocytes, and chondrocytes, and are in part responsible for maintaining tissue integrity. Recently, a progenitor cell population has been found within the synovial fluid that shares many similarities with bone marrow MPCs. These synovial fluid MPCs (sfMPCs) share the ability to differentiate into bone and fat, with a bias for cartilage differentiation. In this study, sfMPCs were isolated from human and canine synovial fluid collected from normal individuals and those with osteoarthritis (human: clinician-diagnosed, canine: experimental) to compare the differentiation potential of CD90+ vs. CD90− sfMPCs, and to determine if CD90 (Thy-1) is a predictive marker of synovial fluid progenitors with chondrogenic capacity in vitro.

Methods

sfMPCs were derived from synovial fluid from normal and OA knee joints. These cells were induced to differentiate into chondrocytes and analyzed using quantitative PCR, immunofluorescence, and electron microscopy.

Results

The CD90+ subpopulation of sfMPCs had increased chondrogenic potential compared to the CD90− population. Furthermore, sfMPCs derived from healthy joints did not require a micro-mass step for efficient chondrogenesis. Whereas sfMPCs from OA synovial fluid retain the ability to undergo chondrogenic differentiation, they require micro-mass culture conditions.

Conclusions

Overall, this study has demonstrated an increased chondrogenic potential within the CD90+ fraction of human and canine sfMPCs and that this population of cells derived from healthy normal joints do not require a micro-mass step for efficient chondrogenesis, while sfMPCs obtained from OA knee joints do not differentiate efficiently into chondrocytes without the micro-mass procedure. These results reveal a fundamental shift in the chondrogenic ability of cells isolated from arthritic joint fluids, and we speculate that the mechanism behind this change of cell behavior is exposure to the altered milieu of the OA joint fluid, which will be examined in further studies.     

The molecular structure and lubricating activity of lubricin isolated from bovine and human synovial fluids.

Lubricin was isolated from bovine ankle, metacarpophalangeal and knee and human knee synovial fluids. The lubricins isolated from the bovine joint fluids had the same amino acid and carbohydrate compositions, but differences were observed in the relative molecular masses. The Mr values of bovine metacarpophalangeal and ankle lubricin determined by light-scattering measurements were about 200 000, whereas values of 132 000 and 143 000 were obtained for the bovine knee lubricin. The human knee lubricin had a similar carbohydrate composition to bovine knee lubricin except for the higher glucosamine content, and the amino acid composition differed slightly. The human sample had a lower glutamic acid content and a leucine/isoleucine ratio of 2:1 compared with 1:1 in the bovine. The Mr value of the human knee lubricin (166 000) was also lower than that of the bovine metacarpophalangeal and ankle samples. The Mr value of the bovine knee lubricin determined by sedimentation-equilibrium measurements was 171 000. The length measurements determined by electron microscopy and also the sedimentation measurements showed considerable polydispersity and indicate that the degree of extension of lubricin molecules can vary. Friction measurements showed that the human knee synovial-fluid lubricin had equivalent lubricating ability in a test system in vitro to that observed for lubricin isolated from normal bovine synovial fluids. The lubricating ability of lubricin was concentration-dependent, and each lubricin sample was able to act as a lubricant in vitro in an equivalent manner to whole synovial fluid at concentrations that are thought to occur in vivo.     

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor): regulation of its production and possible roles in bone metabolism.

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast-like cells. Local bone-resorbing factors such as IL-1, TNF alpha, and LPS strongly induced expression of LIF/D-factor mRNA in both clonal MC3T3-E1 cells and primary osteoblast-like cells. Neither parathyroid hormone nor 1 alpha,25-dihydroxyvitamin D3 stimulated expression of LIF/D-factor mRNA. LIF/D-factor per se did not stimulate expression of its own mRNA. Appreciable amounts of LIF/D-factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D-factor, IL-1, and IL-6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D-factor produced by osteoblasts is in concert with other bone-resorbing cytokines such as IL-1 and IL-6 involved in the bone resorption seen in the joints of RA patients. LIF/D-factor specifically bound to MC3T3-E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D-factor dose-dependently suppressed incorporation of [3H]thymidine into MC3T3-E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D-factor alone had no effect on enzyme activity. These results suggest that LIF/D-factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation with other osteotropic factors.     

Identification of the trypsin-like activity in commercial preparations of eel acetylcholinesterase.

Electricus electrophorus acetylcholinesterase (AChE, EC 3.1.1.7) is reported to possess a trypsin-like activity. We found that purification of AChE removes over 99% of this protease activity, which resides in a single 25 kDa protein with an N-terminal sequence identical to bovine pancreatic trypsin. Digests of neuropeptides using purified eel AChE or bovine pancreatic trypsin gave identical peptide maps. These results indicate that the commercial preparation of eel AChE is contaminated by a trypsin, which is difficult to remove completely during AChE purification.     

Relationship between protease activity and a sialoglycopeptide inhibitor isolated from bovine brain

We have recently described the isolation and purification to homogeneity of a new sialoglycopeptide from bovine brain cell surfaces that reversibly inhibits protein synthesis and DNA synthesis of normal but not transformed cells. Active inhibitory preparations, however, were shown to contain a protease activity that was not lost upon purification. Several experiments were performed to establish the relationship between the proteolytic activity of the sialoglycopeptide and the biological inhibitory activity. Both the protease activity and inhibitory activity were stable at pH 6-8 but were reduced or completely destroyed below pH 4 and above pH 9. Acid inactivation was reversible and upon dialysis, both the biological inhibitory and protease activities were regained. Deglycosylation and CNBr cleavage indicated that the polypeptide backbone, rather than carbohydrate moiety, played an important role in the protease and biological inhibitory activities. Furthermore, chemical modification of amino and tyrosine groups indicated that both residues are essential for both activities. Thus, the biological inhibitory activity and protease activity are very closely related and most likely reside with the same polypeptide sequence.     

Differential expression, function and response to inflammatory stimuli of 11beta-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Expression, activity and function of 11beta-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11beta-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-alpha or IL-1beta (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-gamma was without effect, and there was no difference in 11beta-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production--a characteristic feature of synovial derived fibroblasts--was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11beta-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11beta-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.     

Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

Background

30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).

Results

With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.

Conclusion

Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.     

A novel 17 kD heparin-binding growth factor (HBGF-8) in bovine uterus: purification and N-terminal amino acid sequence

We have purified to near homogeneity a novel 17 kD growth factor from bovine uterus which we designated heparin-binding growth factor-8 (HBGF-8). The growth factor binds tightly to cation exchange resins and to Heparin-Sepharose and is stable to acetone precipitation and labile in acid. Based upon total activity in acetone extracts of bovine uterus stimulating 3H-thymidine incorporation into DNA of serum-starved NIH 313 cells, a 6940 fold purification was achieved with an overall yield of HBGF-8 activity of 0.4%, using extraction of acetone powders and chromatographic separations at neutral pH. Approximately 18 micrograms protein was obtained from 1.2 kg wet weight of tissue. HBGF-8 was clearly separated from 17.5 kD bovine uterus basic fibroblast growth factor (bFGF) by purification and its N-terminal amino acid sequence analyzed. A polypeptide with a unique 25 N-terminal amino acid sequence was found. HBGF-8 was as active as acidic fibroblast growth factor (aFGF) and slightly less active than bFGF in the mouse NIH 3T3 fibroblast mitogenic assay system with an intrinsic specific activity of 5000 dpm/ng under standard assay conditions.