WO1991006573A1 - Inhibitor of laminin neurite promoting activity - Google Patents
Inhibitor of laminin neurite promoting activity Download PDFInfo
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- WO1991006573A1 WO1991006573A1 PCT/US1990/006398 US9006398W WO9106573A1 WO 1991006573 A1 WO1991006573 A1 WO 1991006573A1 US 9006398 W US9006398 W US 9006398W WO 9106573 A1 WO9106573 A1 WO 9106573A1
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- laminin
- neurite
- inhibitor
- promoting activity
- promoting
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- 0 C*C(C1)C2O*C(*[N+](**O*)[O-])*[C@]3(CC*4)C(C)(C)CCC4(*O*C)/C=*1/[C@@]3(C)C2 Chemical compound C*C(C1)C2O*C(*[N+](**O*)[O-])*[C@]3(CC*4)C(C)(C)CCC4(*O*C)/C=*1/[C@@]3(C)C2 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
Definitions
- laminin Components of the basal lamina promote neuritic outgrowth from several types of embryonic neurons in vitro.
- extracellular matrix macromolecules including fibronectin, laminin, collagens type I and IV, and a heparan sulfate proteoglycan have been reported to possess neurite-promoting activity.
- cells release into their culture media neurite-promoting factors that express their activity when bound to polycationic substrata.
- laminin appears to be the most potent factor and it is often found complexed with other extracellular matrix constituents such as proteoglycans and entactin/nidogen.
- These associated molecules affect the behavior of laminin during purification and may alter its character and interaction with extracellular matrix and cell surface components.
- preparations of laminin have been described that have different molecular compositions, immunological properties, heparin binding properties, and proteoglycan affinities.
- laminin's neurite-promoting activity is well documented, there have been no reports of inhibitors of this activity. Such an inhibitor would be useful to study laminin activity as well as a means to control neurite growth.
- the present invention satisfies this need by providing an inhibitor of the neurite promoting activity of laminin.
- the invention provides a purified proteoglycan- associated inhibitor of the neurite-promoting activity of laminin and a purified compound which:
- b) is active whether presented to laminin in solution or after either the inhibitor or laminin is first bound to the culture substratum;
- d) can be prevented from binding to neurite- promoting laminin substrates by polyclonal and some monoclonal anti-laminin or polyclonal anti-entactin antibodies if the laminin contains associated entactin;
- e can be purified from rat RN22 Schwannoma cells
- neurite-promoting activity can be abolished by proteases or glycosaminoglycan lyases but not by heat at 90° for 15 minutes.
- Figure 1 shows DEAE ion-exchange chromatography of 35 S ⁇ -labeled RN22 Schwannoma conditioned medium. Eight liters of unlabeled plus 80 ml of S-labeled conditioned media were applied to a 100-ml DEAE column from which bound material was eluted by a 600-ml 0.15-0.6 M-linear NaCl gradient (represented by a line connecting the onset and completion of the elution) . Fractions (60 X 10 ml) were assayed for total protein ( ⁇ ) , radioactivity (S ) , and laminin immunoactivity in an ELISA using polyclonal rat laminin antiserum ( • ) . The late-running peak of laminin immunoactivity (fractions 30-42, indicated by bar) which overlapped with the SO ⁇ peak was collected and pooled for further fractionation.
- Figure 2 shows further fractionation of the laminin and 35 S-labeled DEAE fraction by a second DEAE ion-exchange step.
- Fractions 30-42 from the first DEAE step were diluted to 0.15 M NaCl and applied to a 6-ml DEAE column from which bound material was eluted in 1-ml fractions by a 60-ml 0.15-0.6 M linear NaCl gradient (represented by a line connecting the onset and completion of the elution) .
- (A) fractions were assayed for radioactivity (Q ) and laminin immunoactivity in an ELISA using a polyclonal antiserum (• ) and monoclonal antibody No. 2E8 ( ⁇ ) • Bars indicate POOL I (fractions 4-12) and
- Figure 3 shows a bioassay of substratum-bound neurite promoting and neurite-inhibiting activities derived from Schwannoma-conditioned medium.
- Purified CG 8 neurons were cultured on polyornithine-coated tissue culture wells pretreated with samples from DEAE ion-exchange (A) POOL I, (B) POOL II, and (C) POOL I and POOL II combined, each diluted 1:10 in a volume of 50 ⁇ l. Phase-contrast photomicrographs were taken after 4 h in culture.
- Figure 4 shows isopycnic centrifugation of the neurite inhibitor.
- POOL II from the DEAE fractionation step shown in Figure 3
- POOL II was made 0.4 M in guanidine-HCl, adjusted to 1.35 g/ml with crystalline CsCl, and then centrifuged.
- Gradient fractions were assayed for 35 S-radioactivity (EJ) # laminin immunoactivity in an ELISA using polyclonal antibodies (# ) , and for the presence of neurite-promoting activity (POOL B) , and inhibitory activity (POOL A) (top horizontal bars) .
- EJ S-radioactivity
- POOL B neurite-promoting activity
- POOL A inhibitory activity
- Figure 5 shows SDS-PAGE of CsCl inhibitory fraction (POOL A) .
- (Lane 1) Aurodye protein staining of the inhibitory material electrophoresed on 4-12% acrylamide gels under reducing conditions and then transferred to nitrocellulose. The inhibitory material was examined by autoradiography before (lane 2) and after digestion with heparitinase (lane 3) or chondroitinase ABC (lane 4) .
- the arrows indicate molecular mass markers: rat yolk sac laminin (400 and 200 kD) , entactin (150 kD) and phosphorylase b (97 kD) .
- Figure 6 shows the inhibition of substratum-bound laminin neurite-promoting activity and recovery of activity after inactivation of inhibitor with heparitinase.
- Polyornithine wells were coated with laminin (50 ng/well) which resulted in maximal neuritic outgrowth by CG 8 test neurons.
- Treatment of the laminin substratum with heparitinase (heparin lyase II) did not alter the activity of laminin.
- the neurite-promoting activity of laminin was inhibited by subsequent treatment with the Schwannoma- derived inhibitor (16 NlU/well) .
- Figure 7 shows that inhibition of a laminin-substratum is localized and persistent.
- the substratum was prepared by treating a polyornithine-coated tissue culture dish with RN22 laminin (500 ng/ml) .
- a narrow strip of inhibitor (CsCl POOL A, 1:100) (arrows) was applied by allowing the inhibitor solution to be drawn across the substratum by capillary action under a fine glass fiber. All incubations were performed in water saturated air to prevent drying.
- Figure 8 shows the effect of application sequences on inhibitory titer.
- Serial dilutions of inhibitor (CsCl, POOL A) and 25 ng of laminin were presented to polyornithine-coated wells in various sequences.
- Inhibitor was presented (a) after laminin, (b) simultaneously with the laminin, (c) preincubated in tubes before their combined presentation to the wells, or (d) before addition of laminin.
- the applied samples were allowed to bind the polyornithine wells for 2 h at 25°C, the wells were washed, the CG 8 neurons seeded and after 4 h the percentage of neurons with neurites greater than four cell body diameters in length was determined. 50-100 neurons were scored per well and two wells were counted per experiment; the data•points represent the mean from four such experiments. Standard deviations were ⁇ 5' .
- proteoglycan-associated inhibitor any molecule or compound which has substantially the same structure and activity as the inhibitor of neurite promoting activity purified from rat RN22 Schwannoma cells as described herein. This inhibitor is likely a proteoglycan but can be any molecule or compound which copurifies with the proteoglycan so long as the neurite inhibitory activity is maintained. Modifications in the structure and function of the inhibitor are also meant to be included as a proteoglycan-associated inhibitor so long as the essential structure is maintained.
- purified 11 is meant the inhibitor is free of many compounds normally associated with or occurring with the inhibitor in its native environment.
- the invention provides a purified proteoglycan- associated inhibitor of the neurite promoting activity of laminin and a purified extract having high affinity for cationic resin, high buoyant density, large heterodisperse appearance on electrophoretic gels, ability to label with inorganic sulfate, sensitivity to trypsin and glycosaminoglycan lyases, heat stability and the ability to inhibit the neurite-promoting activity of alminin.
- the invention also provides a purified compound which:
- b) is active whether presented to laminin in solution or after either the inhibitor or laminin is first bound to the culture substratum;
- d) can be prevented from binding to neurite- promoting laminin substrates by polyclonal and certain monoclonal anti-laminin or polyclonal anti-entactin antibodies if the laminin contains associated entactin;
- e) can be purified from rat RN22 Schwannoma cells; and f) can be abolished by proteases or glycosaminoglycan lyases but not by heat at 90° for 15 minutes.
- a compound which has substantially the same structure and activity as the compound with these characteristics is also provided since the essential structure and function can readily be determined and altered by one skilled in the art to, for example, modify the activity.
- the inhibitors provide a method of inhibiting the neurite-promoting activity of laminin. This method comprises contacting laminin with an inhibitory amount of the inhibitor or the extract.
- this invention specifically provides an inhibitor that can bind to laminin and interfere with its ability to promote neurite outgrowth from cultured neurons.
- This inhibitor has been isolated from medium conditioned by RN22 Schwannoma cells by ion-exchange chromatography followed by isopycnic centrifugation.
- the inhibitory material contains proteoglycan based upon its high affinity for cationic resin, high buoyant density, large heterodisperse appearance on SDS gels, ability to become labeled with inorganic radiosulfate, sensitivity to trypsin and certain glycosaminoglycan lyases, and heat stability.
- the proteoglycan-containing inhibitor obtained from the CsCl density gradient also contained a substantial amount of the sulfated, 150-kD laminin-binding protein, entactin.
- the inhibitory activity can be destroyed by glycosaminoglycan lyase, which results in the reexpression of laminin neurite-promoting activity.
- the laminin can be selectively denatured and its neurite promoting activity destroyed by heat, which apparently causes release of the inhibitor.
- fractionation or treatment that selectively destroys one activity neurite-promotion or inhibition can be expressed independently.
- a membrane fraction from RN22 cells contains a proteoglycan with properties very similar to inhibitor isolated from the conditioned medium (i.e., similar fractionation, appearance by autoradiography, and lyase inactivations) .
- the regulation of proteoglycan synthesis and deposition can dictate whether laminin expresses its inherent neurite-promoting activity.
- the degradation of proteoglycan by specific lyases can remove the inhibition and restore neurite-promoting activity to a previously suppressed substratum.
- laminin neurite-promoting activity can depend as much on a fine tuning of proteoglycan synthesis and degradation as on laminin production and its cell surface or extracellular deposition.
- This discovery provides a method of enhancing the neurite-promoting activity of laminin which is bound to a neurite-promoting inhibitor comprising disassociating laminin from the inhibitor or binding a laminin neurite- promoting inhibitor with a compound which prevents the binding of the inhibitor to laminin.
- the compound which prevents binding can comprise a portion of laminin which is reactive with the inhibitor.
- Laminin preparations contain variable amounts of entactin, a sulfated protein that binds to a region near the cross region of laminin. Since entactin itself can bind laminin, but also copurifies with the inhibitor even through high ionic strength conditions, entactin may have the ability to interact with both laminin and the inhibitor and thus affect inhibitor binding. Anti-entactin antibodies prevent the inhibitor from blocking laminin neurite-promoting activity. Also, the monoclonal antibody 2E8, deposited with the Hybridoma Bank, is known to bind to the cross region of rat laminin and, without diminishing neurite-promoting activity, will protect the neurite- promoting activity of laminin from the inhibitor.
- the ability of the inhibitor to interact with laminin may depend on its association with the cross region of the laminin tertiary structure, a region different from the postulated neurite-promoting domain at the end of the 400- kD long arm.
- the inhibitor may influence the distant neurite-promoting site either directly, by additional binding to another portion of the laminin molecule, or indirectly, by inducing laminin conformational changes.
- the ability of the 2E8 and anti-entactin antibodies to protect the neurite-promoting activity of laminin suggests that entactin or other components may mediate the formation of laminin complexes which modulate the potential neurite-promoting activity of laminin in basement membranes.
- the reagent can be an antibody or peptides analogous to laminin or entactin or the like.
- conditioned media from rat sciatic nerve Schwann cells also contain activity which inhibits neuritic outgrowth in response to laminin.
- the Schwann cell inhibitory activity has properties identical to the RN22 inhibitor.
- RN22 Schwannoma cells Growth conditions for the rat RN22 Schwannoma cells and the methods for labeling with 35 S0 4 have been described within the art. See for example, Davis, G.E. et al., Neurochem. Res. 12:909-921 (1987) for typical growth conditions. Eight liters of RN22 conditioned medium containing 80 ml 35 S-labeled RN22 medium was first applied to a 100 ml bed volume DEAE (DE52; Whatman, Inc., Clifton, NJ) ion-exchange column (2.5 cm-diam) and eluted with a 600 ml linear salt gradient from 0.15-0.6 M NaCl in 10 mM phosphate buffer, pH 8.0.
- DEAE DE52; Whatman, Inc., Clifton, NJ
- Laminin immunoreactivity began to elute at 0.3 M NaCl in the gradient and overlapped with the elution of radiosulfated material. Fractions eluted between 300 and 420 ml of the gradient were pooled and diluted to 0.15 M NaCl with phosphate buffer (10 mM, pH 8.0). This diluted pool was applied to a 10 ml bed volume DEAE column and bound material was eluted using a 60 ml NaCl gradient (0.15-0.6 M) . The laminin immunoreactivity which eluted at 0.2-0.3 M NaCl was collected and pooled (POOL I) .
- the inhibitory activity began to elute at 0.3 M NaCl and was also collected and pooled (POOL II).
- Pool II was concentrated by ultrafiltration using an XM100 membrane (Amicon Corp., Danvers, MA) and dialyzed against 0.4 M guanidine-HCl in 50 mM Tris-HCl, pH 7.5. Solid CsCl was added to give density of 1.35 g/ml and sample were subjected to isopycnic centrifugation at 180,000 g for 48 h. High density fractions (>1.5 g/ml) were pooled (Pool A) and dialyzed against PBS, pH 7.2.
- Laminin antigen was measured by ELISA as described by Engvall, E., Methods Enzymol. 70:419-439 (1980). Polystyrene, 96-well plates were used for coating substrates and the polyclonal and monoclonal antibodies were those described by Davis, G.E. et al., J. Neurosci. 5:2662-2671 (1985). Fractions were assayed for neurite-promoting activity using embryonic day 8 ciliary ganglion neurons. Nurite-promoting assays are well established within the art such as, for example, those described by Manthorpe et al., J. Neurochem. 37:759-767 (1981) and Manthorpe et al., J. Cell Biol.
- the RN22 cells were cultured in the presence of an inhibitor of proteoglycan assembly, 4-methyl umbelliferyl- ⁇ -D-xyloside.
- an inhibitor of proteoglycan assembly 4-methyl umbelliferyl- ⁇ -D-xyloside.
- Conditioned medium from RN22 cultures grown in the presence of 1 mM ⁇ -D-xyloside was collected, heat-treated, and assayed for activity.
- test samples were treated with proteoglycan lyases or proteolytic enzyme before assay for activity or analysis by SDS-PAGE.
- samples were incubated for 3 h at 37°C with heparitinase (Heparin lyase II, Sigma Chemical Co.) at 1 U/ml in 50 mM Tris-HCl, pH 7.5, containing 25 mM sodium acetate and 5 mM calcium acetate and/or chondroitinase ABC (Miles Laboratories, Inc., Naperville, IL) at 0.2 U/ml, under the same conditions except that calcium acetate was excluded. Digestions were terminated by boiling.
- Protease digestion was performed using 100 ⁇ g/ml trypsin (Irvine Scientific, Santa Ana, CA) in PBS, followed by the addition of 200 ⁇ g/ml trypsin inhibitor (Sigma Chemical Co.) Induction and Inhibition of Neurite Outgrowth
- Neurite promotion and inhibition were assayed as substratum-bound activities on polyornithine-coated tissue culture wells (Manthorpe et al., (1983) supra) .
- polyornithine wells were treated for 2 h with purified rat yolk sac laminin purified according to Engvall et al., Arch. Biochem. Biophys. 222:649-656 (1983), or rat RN22 Schwannoma cell conditioned medium fractions in PBS (50 ⁇ l) and then washed with sterile PBS.
- Embryonic day 8 chick ciliary ganglion neurons were seeded (10 3 neurons per well) in serum-free Nl medium (Bottenstein and Sato, Proc. Natl. Acad. Sci. USA 76:514-5517 (1979), containing 1% albumin and ciliary neuronotrophic factor (40 Trophic units/ml) (Barbin et al., J. Neurochem. 43:1468-1478 (1984)), and incubated in 5% C0 2 in humidified air. Neurite outgrowth was scored by phase- contrast microscopy after 4 h by counting the percentage of neurons bearing processes greater than four cell body diameters.
- Neurite-inhibitory activity was assayed by mixing rat laminin and samples to be tested for inhibitory activity (50 ⁇ l total volume) and incubating the mixture in polyornithine wells for 2 h at room temperature, followed by three substrate washes with sterile PBS.
- samples were added as follows: (a) laminin was first bound to polyornithine wells for 2 h followed by washing (the wells were blocked with 1% serum albumin solution in some cases) and then inhibitory sample was added; (b) the inhibitory sample was first incubated in polyornithine wells for 2 h followed by washes and addition of laminin; (c) the inhibitory sample and laminin were preincubated for 1 h in polypropylene tubes before being applied to and incubated in polyornithine wells for 2 h. Ciliary test neurons were seeded, cultured, and scored as described above.
- NIU neurite inhibitory unit
- the laminin antigen (measured by ELISA using polyclonal anti-rat laminin) eluted as two broad and slightly overlapping peaks with approximately 0.2-0.3 M and 0.3-0.4 M NaCl and these peaks contained about one-and two-thirds, respectively, of the recovered antigen.
- the single laminin 35 S0 4 pool from the first column resolved in the second column into two laminin peaks, one of which was associated with nearly all the recovered 35 S-radioactivity.
- the elution of two laminin peaks from the second DEAE column occurred with a little less salt concentration as that of the laminin from the first column. This result suggests that the laminin- 35 S0 4 peak from the first column contained laminin-proteoglycan complexes from which the laminin component can be released with additional fractionation.
- the inhibitory material eluted relatively late from the DEAE column ( Figure 2) and was associated with 35 S0 4 - labeled material, suggesting that it may contain proteoglycan.
- POOL II was submitted to fractionation by isopycnic centrifugation on a CsCl gradient. The CsCl density gradient fractions were examined for radioactivity, laminin immunoreactivity, neurite-promoting, and inhibitory activities. The results are shown in Figure 4. Most of the radio-sulfated material had a high buoyant density (>1.5 g/ml) although a small proportion of radioactivity appeared at a lower density
- the neurite-promoting and inhibitory activities of fractions obtained during various fractionation steps are shown in Table I.
- the inhibitory activity recovered in the high density CsCl fractions (POOL A) represented approximately four times (i.e., 3,800 vs. 1,000 NIUs loaded) the activity found in the native second DEAE POOL II starting material, apparently because laminin (with its neurite-promoting activity) was separated away from the higher density inhibitor. This result shows that the interaction between laminin and inhibitor is reversible.
- CsCl preparation to several treatments followed by bioassay. The results are shown in Table II.
- the inhibitor appears to be heat stable but trypsin sensitive suggesting that protein is important in expression of the inhibitory activity.
- the ability of heparitinase or chondroitinase ABC to eliminate the inhibitory activity suggests the involvement of both heparan and chondroitin sulfate glycosaminoglycans in the inhibitory activity.
- the products of lyase digestion i.e., glycosaminoglycan fragments plus core protein were not active.
- heparin, heparan sulfate, chondroitin sulfate (up to 20 ⁇ g/ml) , or the chondroitin sulfate proteoglycan decorin, which inhibits cell attachment to fibronectin did not decrease the percentage of neurons bearing neurites in response to a laminin substratum.
- Inhibitory sample of CsCl preparation (POOL A, Fig. 4) was treated with heat or with either trypsin (100 ⁇ g/ml) , chondroitinase ABC (0.2 U/ml), or heparitinase (1 U/ml) for 3 h at 37°C.
- conditioned medium from RN22 cultures grown in the presence of methyl-umbelliferyl ⁇ -D- xyloside was collected, heat treated, and assayed for activity.
- medium from ⁇ -D-xyloside-treated cultures contained ⁇ 20% of the inhibitory activity (1.7 and 0.3 NlU/ml, respectively), suggesting that this activity involved an intact proteoglycan.
- the enzyme treatment eliminated the inhibitory activity and restored the neurite-promoting activity of laminin. This shows that the inhibitor does not displace laminin from the substratum but binds to and blocks the substratum-bound laminin.
- a completely inhibited substratum (4 neurite-promoting units [NPU]/well of laminin plus 8 NlU/well inhibitor) was then treated with a high amount of laminin (16 NPU/well) , a neurite-promoting substratum was created (64% ⁇ 14 neurons bearing neurites) .
- the inhibitory pool from the CsCl centrifugation (POOL A, Figure 4) was shown to be free of laminin but yet retained the capacity to interact with laminin and inactivate its neurite-promoting activity.
- This approach used purified RN22 laminin and antibodies that bind to laminin but do not block its neurite-promoting activity. The results are shown in Table III. When RN22 laminin was preincubated with polyclonal antibodies raised against rat yolk sac tumor laminin, which .do not block RN22 laminin activity, its neurite-promoting activity was minimally decreased by the inhibitor.
- pretreatment of the RN22 laminin with 2E8 anti-human laminin monoclonal antibody also prevent inhibition. Since the 2E8 epitope on laminin is located at the cross-region of the laminin molecule, this region of laminin might participate in the interaction between the inhibitor and laminin resulting in inhibition.
- the partially purified inhibitor contains relatively high levels of the laminin-binding protein, entactin, which copurified with the inhibitory proteoglycan even under conditions which separated inhibitory activity from laminin neurite-promoting activity (i.e., isopycnic centrifugation) .
- entactin is known to bind to laminin near the cross-region of laminin (Paulsson, M.M. et al., Eur. J. Biochem. 166:11-19 (1987)).
- Table III Table III. Entactin antibodies, which do not by themselves interfere with the neurite-promoting activity of RN22 laminin (a preparation containing entactin) , did prevent the inhibition from occurring.
- anti-entactin antibodies were shown to be immunoreactive with the inhibitory material as well as with various tested laminin preparations.
- the isolated inhibitory fraction (CsCl Pool A, Figure 4) was fractionated by isopycnic centrifugation run under dissociating conditions (in the presence of 2 M guanidine-HCl) .
- a high buoyant density fraction containing heparan/chondroitin sulfate proteoglycan, but no entactin was obtained that contained approximately 60% of the original inhibitory activity.
- the laminin preparation used in these assays contained entactin immunoreactivity. All preparations of laminin tested contained substantial entactin immunoreactivity and conditions required to dissociate entactin from laminin compromised the neurite-promoting activity of isolated laminin.
- RN22 laminin 25 ng was incubated in 6-mm-diam polyornithine-wells for 2 h.
- the laminin-polyornithine substratum was treated with PBS containing 1% BSA followed by a 30-min incubation with PBS alone or PBS containing 1:50 dilutions of the following antibodies: polyclonal anti-human fibronectin (FN) , monoclonal anti-neurofilament (NF) , polyclonal anti-rat laminin, monoclonal anti-rat laminin No. 2E8, or polyclonal anti-mouse entactin.
- FN polyclonal anti-human fibronectin
- NF monoclonal anti-neurofilament
- polyclonal anti-rat laminin monoclonal anti-rat laminin No. 2E8, or polyclonal anti-mouse entactin.
- inhibitor 160 NlU/ml: CsCl inhibitory fraction
- CG 8 neurons (10 3 /well) were seeded and the percentage of neurons bearing neurites was determined 4 h later by scoring 50-100 neurons per well.
- Conditions were replicated eight times in four separate experiments. Standard deviations were ⁇ 10%.
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Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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NO92921673A NO921673L (en) | 1989-10-31 | 1992-04-29 | INHIBITOR OF LAMININ-NEURITE ACTIVATIVE ACTIVITY |
Applications Claiming Priority (2)
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US42949389A | 1989-10-31 | 1989-10-31 | |
US429,493 | 1989-10-31 |
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WO1991006573A1 true WO1991006573A1 (en) | 1991-05-16 |
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PCT/US1990/006398 WO1991006573A1 (en) | 1989-10-31 | 1990-10-29 | Inhibitor of laminin neurite promoting activity |
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EP (1) | EP0498856A1 (en) |
JP (1) | JPH05504557A (en) |
AU (1) | AU6734190A (en) |
CA (1) | CA2066658A1 (en) |
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US6972168B2 (en) | 2001-08-13 | 2005-12-06 | University Of Florida Research Foundation, Incorporated | Materials and methods for nerve grafting, selection of nerve grafts, and in vitro nerve tissue culture |
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1990
- 1990-10-29 CA CA002066658A patent/CA2066658A1/en not_active Abandoned
- 1990-10-29 AU AU67341/90A patent/AU6734190A/en not_active Abandoned
- 1990-10-29 WO PCT/US1990/006398 patent/WO1991006573A1/en not_active Application Discontinuation
- 1990-10-29 EP EP91900591A patent/EP0498856A1/en not_active Ceased
- 1990-10-29 JP JP2515881A patent/JPH05504557A/en active Pending
Non-Patent Citations (5)
Title |
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Dialog Information Services, File 154: Medline 83-91, NLM accession number 87308367, Carey DJ et al: "Effects of inhibition of proteoglycan syn- thesis on the differentiation of cultured rat Schwann cells" & J Cell Biol Aug 1987, 105 (2) p1013-21 * |
Dialog Information Services, File 154: Medline 83-91, NLM accession number 89007401, Campbell MA et al: "Effects of laminin on attachment, growth anddifferentiation of cultured Y-79 retinoblastoma cells" & Invest Ophthalmol Vis Sci Oct 1988, 29 (10)p1517-22 * |
Dialog Information Services, File 154: Medline 83-91, NLM accession number 89171248, Liesi P et al: "Identification of a neurite outgrowth-pro- moting domain of laminin using synthetic peptides" & FEBS Lett Feb 13 1989, 244 (1) p141-8 * |
Dialog Information Services, File 154: Medline 83-91, NLM accession number 89334896, Sephel GC et al: "Laminin A chain synthetic peptide which supports neurite outgrowth", & Biochem Biophys Res Commun Jul 31 1989, 162 (2) p821-9 * |
Dialog Information Services, File 154: Medline 83-91, NLM accession number 90037230, Muir D et al: "Schwannoma cell-derived inhibitor of the neurite- promoting activity of laminin", & J Cell Biol Nov 1989, 109 (5) p2353-62 * |
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Also Published As
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CA2066658A1 (en) | 1991-05-01 |
AU6734190A (en) | 1991-05-31 |
JPH05504557A (en) | 1993-07-15 |
EP0498856A1 (en) | 1992-08-19 |
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