Patent Application: US-64818297-A

Abstract:
permeability of the blood - brain barrier and other physiological barriers can be modulated by the degree of tyrosine phosphorylation of proteins . agents which promote tyrosine protein dephosphorylation reduce the permeability of the blood - brain barrier and those which promote phosphorylation increase permeability . increasing blood - brain barrier permeability is useful in delivering drugs having a desired effect upon the central nervous system ; decreasing blood - brain barrier permeability and other physiological barrier permeability is useful in preventing undesired compounds reaching the cns and in certain clinical conditions .

Description:
in initial experiments , cells were treated with vanadate , a non - selective , but potent , inhibitor of tyrosine phosphatases . in mdck cells , vanadate alone did not cause a decrease in ter . however , the addition of h 2 o 2 to vanadate pretreated cells caused a rapid decrease in ter ( fig1 a ), indicative of an increased permeability of the tight junctions . vanadate is relatively membrane - impermeant , but h 2 o 2 oxidises vanadate to membrane - permeant pervanadate which then enters the cells to inhibit tyrosine phosphatases ( see eg fantus et al , biochemistry 28 8864 - 8871 ( 1989 ); volberg et al , cell regulation 2 105 - 120 ( 1991 ); o &# 39 ; shea et al , proc . natl . acad . sci . u . s . a . 89 10306 - 10310 ( 1992 )). indeed , preformed pervanadate elicited a decrease in ter ( fig1 b ). over a longer time period , vanadate alone caused a decrease in ter ( fig1 c ), presumably because of its eventual entry into the cells . a more selective inhibitor of tyrosine phosphatases is phenylarsine oxide ( pao ). it inhibits by covalently reacting with closely spaced sulphydryl groups of the phosphatase ( see levenson and blackshear , j . biol . chem . 264 19984 - 19993 ( 1989 ); garcia - morales et al , proc . natl . acad . sci . u . s . a . 87 9225 : 9259 ( 1990 ); pronk et al , j . biol . chem . 267 24058 - 24063 ( 1992 )). it was found that pao could also cause a decrease in ter of mdck strain i cells ( fig2 a ). pao action was blocked by the dithiol , 2 , 3 - dimercaptopropanol , but not by β - mercaptoethanol ( fig2 b ), as expected for pao mediated inhibition via the appropriate configuration of sulphydryls . dimercaptopropanol did not block the decrease in ter elicited by the ca 2 + chelator bis -( o - aminophenoxy )- ethane - n , n , n ′, n ′- tetraacetic acid ( bapta ), indicating selectivity in the inhibition of pao action ( fig2 b ). both reducing agents , however , blocked the action of pervanadate ( fig2 b ), probably by simple reduction of pervanadate to vanadate . the decrease in ter induced by pervanadate ( fig3 a ) or pao ( fig3 b ) could be partially reversed or completely reversed , respectively , by reducing agent if added early enough . thus , tyrosine phosphatase inhibitors elicit a reversible opening of tight junctions in mdck strain i cells . in the bbecs , pervanadate and pao similarly caused a dose - dependent and rapid decrease in ter ( fig4 a to 4 d ). it was previously shown that cyclic amp increases ter , ie decreases leakiness of tight junctions , in the bbecs as well as in endothelial cells derived from human brain ( rubin et al , j . cell biol . 115 1725 - 1735 ( 1991 )). the pervanadate - and pao - induced decrease in ter was observed in bbecs treated with or without cyclic amp ( fig4 a , c with fig4 b , d ). in normal cells , the degree of tyrosine phosphorylation of proteins reflects the balance of the activities of tyrosine kinases and tyrosine phosphatases . pervanadate and pao , by inhibition of tyrosine phosphatases , must cause increased protein tyrosine phosphorylation through active kinases . to investigate the mechanism whereby the phosphatase inhibitors decrease ter , the subcellular localisation of tyrosine phosphorylated proteins was first determined . this was achieved by immunostaining using an anti - phosphotyrosine antibody ( py20 ) ( icn biomedicals , ltd ). in this procedure , the cells are fixed in paraformaldehyde to preserve the organisation and covalent modifications of proteins as found in the intact cell . after membrane - permeabilisation , the fixed cells are incubated with antibody and sites of bound antibody are detected using a flurochrome - conjugated secondary antibody followed by fluorescence microscopy . in both the mdck cells and bbecs , 100 μm pervanadate initially caused an increase in staining at the adherens junction at the intercellular borders . this pattern of staining is similar to that previously reported ( see volberg et al , cell regulation 2 105 - 120 ( 1991 ); volberg et al , embo j . 11 1733 - 1742 ( 1992 )). in contrast , pao ( 10 μm ) caused only an increase in border staining of the mdck strain i cells at times of decreased ter . in the bbecs , pao ( 10 μm ) caused a transient increase in border staining of the cells . these observations indicate that substrates for tyrosine kinases must be present at intercellular junctions . however , these studies give no information as to the identity of the tyrosine phosphorylated proteins . the tyrosine phosphorylation of individual proteins was examined by immunoblotting using an anti - phosphotyrosine antibody . initially , phosphorylation of proteins in whole cell lysates was examined to gauge the selectivity of action of the phosphatase inhibitors . pervanadate caused an increase in the tyrosine phosphorylation of many proteins in both the mdck cells ( fig5 a ) and the bbecs ( fig5 b ). in contrast , concentrations of pao that caused a decrease in ter had much more of a discrete effect , eliciting the phosphorylation of only a few proteins in both the mdck cells ( fig5 c ) and bbecs ( fig5 d ). to determine the identity of proteins that control tight junction permeability , it is necessary to known that tight junctional permeability is known to be influenced by the integrity of the adherens junction . removal of extracellular ca 2 + leads to an increase in the permeability of the tight junction presumably by disruption of the adherens junction ( see gumbiner et al , j . cell . biol . 107 1575 - 1587 ( 1988 )). the ca 2 + - sensitive components of the junctional complex are the cadherins . these transmembrane proteins mediate intercellular adhesiveness in a ca 2 + - dependent , homophilic manner via their extracellular domain . the cytoplasmic domain of the cadherins associates with three further proteins termed α -, β - and γ - catenins , which link the cadherins to the cytoskeleton ( stappert and kemler , curr . opinion in neurobiol . 3 60 - 66 ( 1993 )). recently , it was reported that pervanadate treatment or pp60 v - scr over - expression resulted in the tyrosine phosphorylation of components of the cadherin / catenin complex ( matsuyoshi et al , j . cell biol . 118 703 - 714 ( 1992 ); behrens et al , j . cell biol . 120 757 - 766 ( 1993 ); hamaguchi et al , embo j . 12 307 - 314 ( 1993 )). such increased phosphorylation was associated with decreased cadherin - dependent cell adhesiveness ( matsuyoshi et al , j . cell biol . 118 703 - 714 ( 1992 ); behrens et al , j . cell biol . 120 757 - 766 ( 1993 ); hamaguchi et al , embo j . 12 307 - 314 ( 1993 )). it is possible that pervanadate and pao increase the permeability of tight junctions by causing the tyrosine phosphorylation of components of the cadherin / catenin complex . it was therefore decided to examine more closely the effect of pervanadate and pao on the tyrosine phosphorylation of this complex in mdck strain i cells . the complex was immunoprecipitated from cells solubilised in a way that preserves pre - existing protein interactions using rrl , a mouse monoclonal antibody that recognises e - cadherin ( gumbiner et al , j . cell biol . 102 457 - 468 ( 1986 )). immunoprecipitated proteins were separated by sds - page , and tyrosine phosphorylation was detected by immunoblotting using an anti - phosphotyrosine antibody and enhanced chemiluminescence . ( i ) β - catenin , not α -, appears to be phosphorylated in response to pao pervanadate caused an increase in the tyrosine phosphorylation of several proteins in anti - e - cadherin immunoprecipitates with molecular means expected of e - cadherin ( 120 kda ) and associated catenins ( molecular mass range , − 80 - 102 kda : fig6 a ). however , pao predominantly stimulated the tyrosine phosphorylation of only one of the catenins ( fig6 a ), which on the basis of apparent molecular mass could be α - or β - catenin . to address the issue of the identity of the catenin phosphorylated in response to pao , peptide - directed antibodies were raised that specifically recognize α - or β - catenin . pao - treated cells were lysed in sds , followed by heating to dissociate protein complexes . under these conditions only individual tyrosine phosphorylated proteins , not proteins associated with tyrosine phosphoproteins , are immunoprecipitated using anti - phosphotyrosine antibody . in such immunoprecipitates , β - catenin is rapidly increased in response to treatment of the cells with either pao or , as expected , pervanadate ( fig6 b ). however , α - catenin was not detectable in the phosphotyrosine immunoprecipitates ( fig6 c ). moreover , pao - or even pervanadate - stimulated tyrosine phosphorylation of α - catenin could not be detected ( fig6 e ) in α - catenin immunoprecipitates ( fig6 d ). thus , the tyrosine phosphorylation of β - catenin phosphorylation is increased in response to pao , accounting for its immunoprecipitation by phosphotyrosine antibody . furthermore , this increased phosphorylation appears to be rather selective in that α - catenin does not appear to be tyrosine phosphorylated . since the effects of pao on ter could be reversed by reducing agents , it was determined whether catenin phosphorylation was similarly reversed . cells were treated for 15 minutes with pao to stimulate tyrosine phosphorylation of the catenin , and then treated with or without 2 , 3 - dimercaptopropanol for a further 45 minutes , ( fig3 ). the tyrosine phosphorylation of the catenin observed at 15 minutes persisted for up to 60 minutes ( fig6 f ). however , addition of 2 , 3 - dimercaptopropanol reversed catenin tyrosine phosphorylation as content of the immunoprecipitates was relatively invariable ( fig6 g ), indicating quantitatively successful immunoprecipitation of the complex under all conditions . thus , the reversible decrease in ter induced by pao correlated with reversible catenin tyrosine phosphorylation . although tight junction permeability is known to be dependent on the adherens junction , and it is shown using pao that catenin phosphorylation correlates with the decrease in ter , it is possible that tight junction - associated proteins may also be substrates for tyrosine kinases . therefore , the possibility that tight junction associated proteins in mdck cells were tyrosine phosphorylated in response to pao was examined . zo - 1 and its associated protein zo - 2 were immunoprecipitated using an anti - zo - 1 antibody and tyrosine phosphorylation was examined by immunoblotting . pao clearly stimulated the tyrosine phosphorylation of zo - 1 and to a lesser extent that of zo - 2 ( fig7 ). pervanadate clearly resulted in the tyrosine phosphorylation of zo - 1 , zo - 2 and , to a much lesser extent , that of a protein of 130 kda ( fig7 ), possibly the same protein as that identified by balda , m . s ., gonzalez - mariscal , l ., matter , k ., cereijido , m . and anderson , j . m ., 1993 , assembly of the tight junction ; the role of diacylgycerol . j . cell biol . 123 : 293 - 302 . these data illustrate that tight junction proteins as well as catenins are phosphorylated in response to pao , raising the possibility that modulation of tight junction permeability could be achieved , either directly or indirectly , via changes in adherens junction adhesiveness and / or by direct modulation of tight junction permeability . further data is provided later on which shows that certain proteins which are associated with the cadherin / catenin complex can also be tyrosine phosphorylated . 2 . the tyrosine kinase inhibitor herbimycin a attenuates the effects of pao pretreatment of mdck cells with herbimycin a attenuated the decrease in transcellular electrical resistance elicited by subsequent addition of pao ( fig8 ). herbimycin a attenuated the ability of pao to increase protein tyrosine phosphorylation ( fig9 ), correlating with its effects on tight junction permeability ( fig8 ). this inhibitory effect of herbimycin a is consistent with pao action via inhibition of tyrosine phosphatases and suggests that a tyrosine kinase inhibitor could be useful in modulation of tight junction permeability . 3 . anti - phosphotyrosine label of rat brain endothelial cells after middle cerebral artery ( mca ) occlusion stroke is the result of reduced blood supply causing ( usually ) focal ischaemia in the brain . this can be caused by either the rupture of a blood vessel ( haemorrhagic stroke ) or due to blood vessel blockage by a blood clot ( occlusive stroke ). recovery from stroke is compromised by development of oedema that begins soon after the initial insult , and continues for several days . this oedema , if widespread , causes increased intracranial pressure and can harm the brain , reducing the extent of recovery . the oedema seen in the first hours after stroke is likely to be cytotoxic , caused by swelling of cellular elements in the region of damage . however , oedema is still present for the next few days and this maintained oedema is vasogenic , i . e . the result of increased permeability of brain capillary endothelial cells . it is likely that the blood vessels with increased permeability are microvessels supplied by a different artery adjacent to the area of infarct , and it is possible that the endothelial cells liming these vessels respond to locally released factors from ischaemic tissue nearby . in this case agents that interfere with the action of these locally released factors are of potential therapeutic value in that they could prevent the loss of blood - brain barrier integrity , ameliorate that prolonged oedema after stroke and improve the extent and speed of recovery of the patient . in humans , the most frequently encountered stroke syndrome is associated with infarction in the area of the territory of the middle cerebral artery ( mca ). the mca supplies blood to a large area of the cortex through surface branches and to several deeper structures through smaller penetrating branches . a commonly used in vivo model of severe stroke is mca occlusion in adult rats . here , focal cerebral ischaemia was induced by permanent occlusion , by cauterization , of the left mca of adult sprague dawley rats . after 24 hours , rats were either sacrificed with no further operation or were intravenously injected with evan &# 39 ; s blue dye ( see below ) 30 minutes before being sacrificed . the brain was removed immediately and frozen . the area of brain served by the mca was visibly swollen 24 hours after occlusion . evan &# 39 ; s blue : the blood protein albumin is restricted to the lumen of vessels that have an intact blood - brain barrier , but when blood - brain barrier function is lost albumin leaves the vessels and enters the brain parenchyma . evan &# 39 ; s blue dye binds to albumin in the blood , and when albumin leaks into the tissues albumin - associated dye can be detected by fluorescence microscopy . frozen sections of unfixed tissue were examined for evan &# 39 ; s blue extravasation into the brain parenchyma . mca occlusion caused evan &# 39 ; s blue to leak into the brain parenchyma in part of the left cortex , this could be detected within 24 hours . gross morphological changes in the labelled area indicated that the brain was severely damaged following the operation . tissue away from the damaged area ( e . g . in the right cortex ) did not label with evan &# 39 ; s blue . albumin : blood - brain barrier breakdown was also detected by incubating sections with antibody to rat albumin to demonstrate albumin leakage . sections of brain labelled in this way also showed increased labeling of the parenchyma ( outside the vessels ), near the site of damage , whilst undamaged areas were again unlabelled , indicating a local loss of blood - brain barrier function at the sites affected by occlusion . ( iii ) effects of mca occlusion on blood - brain barrier capillary endothelial cells as discussed previously , control of the level of protein tyrosine phosphorylation appears to be essential for the maintenance of functional tight junctions . many ( bioactive ) factors act through receptors that can alter levels of tyrosine phosphorylation , either through changing the activity of tyrosine kinases or tyrosine phosphatases . if factors released during ischaemia increase endothelial cells permeability by a mechanism that depends on alteration of the level of protein tyrosine phosphorylation , then it is possible that their action could be controlled through drugs that prevent elevation of protein tyrosine phosphorylation . the advantage of this approach is , firstly , that if the increased permeability is caused by many factors acting through different pathways that converge at the control of tyrosine phosphorylation levels , then one drug could be used to control the action of many released factors . secondly , effective treatment would not need to await the identification of the active agents and the subsequent development of specific antagonists . in these experiments , brain sections were labelled with an antibody to phosphotyrosine ( monoclonal antibody 4g10 ) and labelled with a polyclonal antibody to collagen type iv to outline blood vessels . in sham operated animals , 4g10 label was restricted to a few large , peripherally located , vessels with a pattern of staining consistent with tyrosine phosphorylation of proteins at the borders of cells lining the vessels . after 24 hours of mca occlusion an increase in 4g10 label was seen associated with microvessels located at the edge of the severely damaged tissue ( see fig1 ). the increase in 4g10 label was restricted to vessels in the area of the lesion , but not every vessel was labelled . 4g10 distribution in the microvessels is similar to that seen when brain tissue from normal adult rat is exposed to pervanadate , a drug that elevates levels of phosphotyrosine by inhibiting the enzymes responsible for tyrosine dephosphorylation . however , pervanadate induces increased phosphotyrosine in both small and large blood vessels , whilst after 24 hours of mca occlusion , only microvessels showed increase phosphotyrosine label . in control experiments , 4g10 binding was prevented by perincubation of the antibody with phosphotyrosine indicating specific binding of 4g10 to phosphotyrosine . the above data suggests that the level of tyrosine phosphorylation of proteins at brain microvessel endothelial cell borders is upregulated 24 hours after mca occlusion . it is likely that blood - brain barrier leakiness seen after stroke is caused by release of factors from ischaemic tissue that act by elevation of endothelial cell protein tyrosine phosphorylation . drugs that prevent phosphotyrosine elevation could reduce the loss of blood - brain barrier integrity that causes oedema following stroke , and hence prevent some of the ensuing permanent damage to neuronal cells . in addition to the particular catenins discussed above it has also been found that other proteins — p100 and p120 — which are associated with the catenin cadherin complex are tyrosine phosphorylated . these are discussed below : immunoblot analysis of a variety of different cells revealed that the anti - p120 monoclonal antibody 2b12 ( kanner et al ., 1990 ) recognized a broad band of approximately 120 kda . another anti - p120 monoclonal antibody that had been raised against the c - terminal portion of p120 recognized the same as 2b12 and , in addition , another cluster of bands at 100 kda . the reason for the multiplicity of bands at 120 kda and 100 kda is not clear , but obviously they are immunologically related . the pattern of appearance of these multiple bands also depended on the cell type . for example in mdck cells , the bands were very diffuse , whereas in mdbk cells they were clearly resolved , especially when visualized by [ 35 s ] methionine labelling in the absence of fluorographic reagent . it is possible that post - translational modification , such as phosphorylation and myristoylation may account for the appearance of multiple bands . the relationship , apart from the immunological one , between p120 and p100 is not yet clear . the northern blots described by reynolds et al . ( 1992 ) suggested the possibility of p120 - related gene products or alternatively spliced transcripts . furthermore , southern analysis apparently indicated that one or more p120 - related genes exist ( reynolds et al ., 1992 ). it follows that the cluster of bands corresponding to p120 and the similar cluster to p100 could also represent splice variants of , respectively , p100 and p120 . it is also possible that p100 could simply represent a degradation product of p120 , although samples were prepared in denaturing buffer and immunoprecipitations were performed in the presence of inhibitors of a broad spectrum of proteases . our results provide evidence that p120 / p100 associates with the cadherin / catenin complex . thus , from tx - solubilized epithelial cells , anti - p120 antibody immunoprecipitated [ 35 s ] methionine - labelled proteins that comigrated with those immunoprecipitated by cadherin antibodies ( fig1 ). immunoblot analysis verified the identity of these proteins as cadherins and catenins ( fig1 ). similarly , anti - p120 reactive protein was seen in the anti - e - cadherin immunoprecipitates from mdck cells , although on the basis of mobility this appears to represent primarily p100 , not p120 ( fig1 ). however , it should be noted that p120 immunoreactive protein was not unequivocally identified in the mdck cells because of the poor reactivity of the 2b12 antibody with canine protein . this apparent association between p120 / p100 and the cadherin / catenin complex was not restricted to epithelial cells as similar results were obtained using endothelial cells , both by [ 35 s ] methionine - labelling ( fig1 ) and immunoblot analysis ( fig1 ). however , although our results clearly show an interaction between catenins and p120 / p100 in endothelial cells , we did not identify a cadherin in the anti - p120 immunoprecipitates . the interpretation of the biochemical analyses of the protein complexes in the epithelial and endothelial cells is further supported by the immunocytochemical study . in cultured cells and in tissue sections , anti - p120 immunoreactivity was strongly associated with intercellular junctions , displaying a localization that was remarkably similar to that obtained with anti - β - catenin antibody ( fig1 and 18 ). here , we were restricted to using the anti - p120 antibody which might recognize either p120 or p100 , or both . of course , one explanation for the ability of anti - p120 antibody to immunoprecipitate the cadherin / catenin complex is that it cross - reacts with one of the known components of the complex . however , this is unlikely for the following reasons . tds lysis of cells dissociates α - catenin and γ - catenin from the cadherin and β - catenin complex ( see mccrea and gumbiner , 1991 ). in anti - e - cadherin immunoprecipitates from mdck cells lysed in tds buffer , anti - p120 antibody failed to react with e - cadherin or the co - precipitating β - catenin . in anti - α - catenin immunoprecipitates from mdck cells lysed in tds buffer , anti - p120 antibody failed to react with α - catenin ( results not shown ). as shown in fig1 , β - catenin immunoprecipitates from tds - lysed endothelial cells , although clearly containing β - catenin , and presumably a cadherin , did not contain any anti - p120 reactivity . cross - reactivity with γ - catenin , a protein of approximately 85 kda , is unlikely as the immunoblots shown in fig1 fail to show reactivity with protein below 100 kda . one protein that has to be addressed concerns the lack of any obvious [ 35 s ] methionine - labelled bands that correspond to p120 / p100 in the cadherin immunoprecipitates from tx lysed cells . thus , as shown in fig1 , rr1 and the anti - p120 immunoprecipitates from mdck cells look remarkably similar . however , it is clear from the anti - p120 immunoprecipitates from tds lysed cells that the anti - p120 reactive material migrates as a broad band , the bulk of which migrates between α - and β - catenin , with the remainder migrating slightly slower than α - carenin . in conjunction with the immunoblot analysis shown in fig1 , which demonstrates that only a fraction of the anti - p120 immunoreactive material is immunoprecipitated by rr1 , and the fact that p120 contains about half of the number of methionenesin α - and β - catenin , if steady - state labelling is assumed , then it is obvious that it would be difficult to see any labelling corresponding to the anti - p120 immunoreactive material in the rr1 immunoprecipitates . similarly , with respect to the experiments with the brain endothelial cells ( fig1 ), under the extraction conditions employed it appears that only a fraction of the pool of the cadherin / catenin complex associates with p100 , and even less with p120 . thus , it would be difficult to see labelling of bands corresponding to p120 and p100 in the β - catenin immunoprecipitates as this region of the gel is already occupied by other major brands . it is also difficult to discern a band corresponding to the cadherin seen in the β - catenin immunoprecipitates in either the anti - p120 or 2b12 immunoprecipitates because of the dominance of the labelling of p120 ( fig1 ). it is emphasized that there are also clear quantitative differences with respect to the ability of 2b12 and anti - p120 antibody to immunoprecipitate the cadherin / catenin complex . due to poor reactivity of 2b12 with canine protein , this could not be addressed with mdc cells . in bovine brain endothelial cells , the anti - p120 antibody immunoprecipitated the cadherin / catenin complex , although not as well as anti - β - catenin antibody . 2b12 was even less effective than anti - p120 . as anti - p120 recognizes p120 and p100 , and 2b12 only p120 , the difference in the amount of the complex immunoprecipitated must be attributable to greater association of p100 with the catenins . as far as we can tell , the efficiency of anti - p120 and 2b12 in immunoprecipitation was similar in this experiment ( see fig1 for the similar intensity of a band corresponding to p120in the anti - p120 and 2b12 immunoprecipitates from cells lysed in tx buffer ). these data also indicate that independent complexes of p100 and p120 exist with catenins , rather that a catenin / p100 / p120 complex . if the latter were the case , 2b12 would be expected to immunoprecipitate as much of the catenins as anti - p120 antibody . our early study provides a link between p120 / p100 and adherens junction proteins . it is possible that such an interaction , perhaps via the influence of regulatory kinases , such as src , lyn and yes ( see tsukita et al ., 1991 ), may play a role in the modulation of cadherin function , and thereby other cellular functions influenced by the adherens junction . with respect to phosphorylation , the tyrosine phosphate inhibitor phenylarsine oxide was found to cause an increase in tight junction permeability in mdck cells ( staddon et al ., j . cell sci . in press ). this inhibitor increased the tyrosine phosphorylation of the anti - p120 immunoreactive material ( a major p100 band , a minor p120 band ) in these cells , as analyzed by anti - p210 immunoblotting of anti - p120 immunoprecipitates from sds lysates ( results not shown ; see staddon et al ., j . cell sci . in press ). the p120 / p100 proteins could also be involved in the interaction between cadherins and the actin - based cytoskeleton , p120 / p100 may also be part of a signaling cascade , communicating information about the state of cell - cell adhesiveness to the interior of the cell . β - catenin is an arm protein ( mccreas et al ., 1991 ) and can associate with cadherins and the apc gene product ( rubinfeld et al ., 1993 ; su et al ., 1993 ), also an arm protein ( see peifer et al . 1994 ). p120 is an arm protein ( reynolds et al ., 1992 ; peifer et al ., 1994 ), and , as we describe here , p100 is an immunologically related protein . these proteins can interact with β - catenin . the exact nature of the interaction between p120 / p100 and the catenins remains to be established . these proteins may interact directly , or associate with different regions of the cytoplasmic domain of cadherins . other linking or intermediary binding proteins could also be involved . clearly , there appears to be diverse interactions among arm proteins , suggesting the importance of the arm motif in intracellular signailing . in summary , our studies identify p100 and p120 - related protein . we present evidence that these proteins interact with the cadherin / catenin complex . given the important role of the cadherin / catenin complex in cellular transformation and the identification of p120 as a pp60 src substrate , this suggests that p120 / p100 may play a role in cellular growth control and other processes . such as tight junction permeability control , via an influence on cell - cell adhesion . other groups have also observed increased tyrosine phosphorylation of components of the cadherin / catenin complex , but under conditions that differ from those described here . in pp60 v - src transfected rat 3y1 cells , a fibroblast cell line , vanadate induced the tyrosine phosphorylation of e - or p - cadherin and β - catenin ( matsuyoshi et al , j . cell biol . 118 703 - 714 ( 1992 )). in chicken embryo fibroblasts , pp60 v - src caused the tyrosine phosphorylation of n - cadherin and α - and β - catenin . pp60 v - src activity in mdck cells resulted in a rapid ( 10 - 30 min ) tyrosine phosphorylation of e - cadherin and β - catenin ( behrens et al , j . cell biol . 120 757 - 766 ( 1993 )). behrens et al ( 1993 ) also reported that pp60 v - src resulted in a decrease in ter of mdck cells . however , the decrease in ter was observed several hours after tyrosine phosphorylation of e - cadherin / β - catenin . furthermore , pp60 v - src resulted in a major alteration in cell morphology , from epithelial to fibroblast - like . the results of behrens et al ( 1993 ) differ from the present results in that a very rapid decrease in ter is shown here in the absence of any gross changes in cell morphology . our data suggest that the tyrosine phosphorylation of components of the cadherin / catenin complex ( particularly β - catenin ) , and / or of certain proteins associated with this complex ( particularly p120 and a protein referred to herein as “ p100 ”) , may serve to regulate acutely the permeability of the tight junction . thus , intracellular kinase - mediated tyrosine phosphorylation , like the removal of extracellular ca 2 + , may led to a rapid uncoupling of the adherens junction . consequently , this leads to an opening of the tight junction . the conserved cytoplasmic domain of cadherins is known to associate with three proteins , termed α -, β - and γ - catenin ( ozawa et al ., 1989 ), which serve to link cadherins to the actin - based cortical cytoskeleton ( hirano et al ., 1987 ). the association of cadherins with catenins is essential for intercellular ca2 +- dependent adhesiveness ( nagaruchi and takeichi , 1988 ; ozawa et al ., 1991 ; kintner , 1992 ). α - catenin is homolgous to vinculin ( herrenknect et al ., 1991 ; nagafuchi et al ., 1991 ), making it a good candidate for interaction with the actin - based cytoskeleton ( see ozawa et al ., 1990 ; hirano et al ., 1992 ). β - catenin is homologous to the drosophila segment polarity gene armadillo , suggesting a role in developmental signalling in vertebrates ( mccrea et al ., 1991 ), γ - catenin is probably identical to plakogiobin ( knudsen and wheelock , 1992 ; but see piepenhagen and nelson , 1993 ), which again is homologous to armadillo ( see franke et al ., 1989 ; peifer and wieschasu , 1990 ). indeed , β - catenin and plakoglobin appear to form a multigene family ( peifer et al ., 1992 ). a repeating 42 amino acid motif that was originally identified in armadillo ( riggleman et al ., 1989 ) has also been found in several other proteins , including β - catenin and plakoglobin , with a variety of functions ( peifer et al ., 1994 ). these include the apc gene product , a tumour suppressor protein ( kinzler et al ., 1991 ), p120 , a pp60 src substrate ( reynolds et al ., 1992 ), smggds , an exchange factor for ras - related g proteins ( kikuchi et al ., 1992 ), a suppressor of rna polymerase i mutations in yeast ( yano et al ., 1192 ; 1994 ) and band 6 protein , a major desmosomal constituent ( hatzfeld et al ., 1994 ). the function of the repeats in these arm proteins is unknown . interestingly , the apc gene product associates with β - catenin ( rubinfeld et al ., 1993 ; su et al ., 1993 ), supporting an important role for catenins in intracellular processes that regulate cell growth . furthermore , this illustrates that cadherins are not exclusive cellular partners of catenins , raising the possibility of other interactions among catenins , cadherins and arm proteins , important in a variety of biological processes . p120 was initially identified as one of several substrates of the tyrosine kinase pp60 stc ( reynolds et al ., 1989 ; kanner et al ., 1990 ). it is membrane - associated and can be myristoylated , but does not appear to be glycosylated ( kanner et al ., 1991 ). mutational analysis suggested that tyrosine phosphorylation of p120 is necessary for that of pp60 stc - mediated cellular transformation ( linder and burr . 1988 ; reynolds et al ., 1959 ). tyrosine phosphorylation of p210 was also observed in response to epidermal growth factor , platelet - derived growth factor , colony - stimulating factor 1 and in polyoma virus middle t antigen - transformed cells ( downing and reynolds , 1991 ; kanner et al ., 1991 ), but the exact role of p120 in cellular physiology and pathology remains to be established . our data suggest that the permeability of tight junctions is crucially dependent upon the balance of tyrosine kinase ( s ) and tyrosine phoslohatase ( s ) that determine the phosphorylation of the cadherin / catenin complex and / or of certain proteins associated therewith . appropriate stimulation of a tyrosine kinase ( or inhibition of tyrosine phosphatases ) should , therefore , open up the blood - brain barrier or other barrier involving tight junctions . conversely , inhibition of endothelial cell tyrosine kinase ( or activation of tyrosine phosphatases ) would lessen , say , the amount of oedema that results from increased blood - brain barrier leakiness following stroke and lessen or prevent cedema associated with brain tumours . similarly , during situations involving cell trafficking across the blood - brain barrier , inhibition of endothelial tyrosine kinase should prevent cell migration . available tyrosine kinase inhibitors include : genistein , herbimycin a , lavendustin a ; methyl 2 , 5 - dihydroxy - cinnamate ; staurosporine and tyrphostins . tyrosine kinase activators include various ligands , such as fgf , pdgf and vegf , which activate receptors that couple to appropriate tyrosine kinases . also , it may be that , during ischaemia , a ligand is released from glial or neuronal cells which activates an endothelial cell tyrosine kinase , thereby causing opening of the blood - brain barrier ; a blocker of this ligand would be particularly useful in stroke therapy . tyrosine phosphatase inhibitors include vanadate and phenylarsine oxide , of which the latter is preferred because of its more specific activity . agents which promote tyrosline protein dephosphorylation , and therefore promote closure of a physiological barrier such as the blood - brain barrier are useful in a number of ways . such a medicament may be useful in decreasing brain oedema following stroke or associated with brain tumours and / or in blocking the entry into the brain of both leukocytes that mediate an immune response , such as occurs in multiple sclerosis , and metastatic cancer cells that may form tumours , such a medicament may also be useful in promoting tyrosine protein dephosphorylation in peripheral endothelial cells to prevent or mitigate peripheral oedema such as high altitude pulmonary oedema . a further use for such a medicament would be in promoting tyrosine protein dephosphorylation in gastric epithelial cells to treat gastric ulcer , which may be exacerbated by loose tight junctions ( ohkusa et al . gut 34 86 - 89 ) 1993 )). agents which promote tyrosine protein phosphorylation , and therefore promote opening of a physiological barrier such as the block - brain barrier are also useful in a number of ways . such a medicament may be useful in promoting peripheral endothelial cell tyrosine protein phosphorylation to increase delivery of chemotherapeutic agents to peripheral tumours . ( although peripheral tumours do not have a barrier as tight as the blood - brain barrier , the centre of the tumour is often less well vascularised .) such a medicament may also be useful in promoting pulmonary epithelial cell tyrosine protein phosphorylation , preferably when the medicament is administered using an inhaler , so that tight junctions become leaky to fluid , which can then act to dilute the accumulation of mucous in the airways . according to a third aspect of the invention , there is provided the use of an agent which promotes tyrosine protein phosphorylation and the use of a blood - brain barrier - or other physiological barrier - impermeant drug in the preparation of a medicament for delivering the drug to the brain or other part of the body across the barrier . the drug may be any pharmaceutically , veterinarily or diagnostically useful compound or composition of compounds which is normally impermeant to the block - brain or other physiological barrier or at least insufficiently permeant . the pharmacological nature of the drug is otherwise unimportant . the invention is thereofore useful in the delivery of a wide range of drugs across physiological barriers such as the blood - brain barrier . however , it is anticipated that among the primary candidates for delivery by means of this aspect of the invention will be : anti - tumour compounds , such as methotrexate , adriamycin and cisplatin ; growth factors , such as ngf , rdnf and cntf , which are used to treat neurodegenerative disease ; imaging agents , especially those that are antibody based ; and neurotransmitter antagonists or agonists which do not penetrate the blood - brain barrier ( such as certain nmda receptor blockers ). according to a fourth aspect of the invention , there is provided a composition comprising an agent which promotes tyrosine protein phosphorylation and a drug to be delivered across a physiological barrier such as the blood - brain barrier . the composition may also contain a pharmaceutically or veterinarily acceptable carrier . in addition to being useful in the delivery of drugs across physiological barriers such as the block - brain barrier , the invention may also useful in preventing certain unwanted drugs crossing the barrier , if their transit across the barrier is by way of tight junctions . compositions of , and agents useful in , the invention may be adapted for administration by any suitable means , enteral or parenteral . compositions for parenteral administration , such as injection or infusion , will generally be sterile . the dosage of any particular agent will depend on a number of factors and is likely to be optimised in experimental or clinical trials . in any event , the appropriate dosage for a particular patient or subject will be determined by the responsible physician or clinician in each case . preferred features of each aspect of the invention are as for each other aspect , mutatis mucandis . the invention will now be illustrated by way of example only . in the following examples the materials and methods discussed below are utilised . tissue culture - treated , polycarbonate transwell filters ( 0 . 4 μm ) were from costar . all tissue culture materials were from gibco except for plasma - derived horse serums which was from lamoire biological laboratories ( pipersville , pa .). vanadate was from sigma and phenylarsine oxide ( pao ) was from kodak , gel electrophoresis reagents were from bio - rad . other reagents used were of the highest grace commercially available . antibodies against α - and β - catenin were raised in rabbits using synthetic peptides coupled to keyhole limpet haemocyanin ( pierce ) by glutaraidehyde . the peptide for α - catenin corresponded to amino acids 890 - 901 of mouse αe - catenin ( herrenknecht et al ., 1991 ), the pepdde for β - catenin to amino acids 751 - 781 of mouse β - catenin ( butz et al ., 1992 ). the β - catenin peptide contained an addition c - terminal lysine for coupling , which is not in the protein . after several rounds of intradermal and subcutaneous immunisations in intervals of 3 weeks using the ribi adjuvant system ( ribi immunochem research , inc . ), specific antibodies were isolated by affinity chromatography on peptide - ε - carboxyhexanoyl ( ech )- sepharose ( pharmacia ) columns ( 6 and 15 mg of α - catenin and β - catenin peptide , respectively , coupled to 1 of ech - sepharose 4b as described by pharmacia ). the rat monoclonal and - zo - 1 antibody ( r40 . 76 ) was from dr . bruce steveruon ( department of anatomy and cell biology , university of alberta , edmonton , canada ). the mouse monoclonal anti - e - cadherin antibody rr1 was from dr . barry gumbiner ( memorial sloan - kettering cancer center , new york , n . y .). rat monoclonal anti - α - catenin ( α - 18 ) was from prof . shoichiro tsuldta and dr . akira nagaruchi ( department of information physiology , national institute for physiological sciences , okazaki , japan ). monoclonal anti - phosphotyrosine antibody py20 was from icn biomedicais . horseradish peroxidade - conjugated anti - phosphotyrosine antibody rc20 h was from tramduction laboratories . phosphotyrosine antibody ( 4g10 ) conjugated to agarose was from upstate biotechnology inc . fitc - conjugated goat anti - rat and anti - mouse igg were from jackson immunoresearch laboratoried ltd . all cells were maintained at 37 ° c . under 5 % co 2 in humidified air . mdck cells ( strain 1 ), from dr . barry gumbiner , were maintained in mem containing 10 % fcs . for experimental use , 10 5 cells in 0 . 2 ml of medium were plated on 6 . 5 mm transwell filters . the basolateral chamber contained 0 . 75 ml of medium . the cells were used between 4 and 7 days after plating . primary cultures of bovine brain endothelial cells were prepared essentially as described by rubin et al . ( 1991 ). the cells were continuously grown in 30 % astrocyte - conditioned medium . after plating 2 . 5 × 10 4 cells on 6 . 5 mm , collagen - coated transwell filters , the cells were maintained for 2 - 3 days in the absence or presence of 250 μm 8 -( 4 - chlorophenylthio ) camp ( cpt - camp ) plus 17 . 5 μm of the phosphodiesterase inhibitor ro20 - 1724 ( calbiochem ), as described by rubin et al . ( 1991 ). for experiments involving mdck cells , culture medium was replaced with mem containing 0 . 5 % calf serum for at least 2 hours prior to use . with the brain endothelial cells , additions were made directly to the medium in which the cells were cultured . initial resistance measurements were taken using a millicell - ers resistance system ( millipore ). aqueous solutions were added to the apical and basolateral chambers of the transwell from 100 - fold concentrated stock solutions . vanadate was prepared as a 0 . 1 m stock solution , ph 7 . pao was dissolved in dmso and added directly to the medium from a 1000 - fold concentrated stock . resistance measurements were subsequently taken as indicated . in all cases , appropriate solvent controls had no effect on resistance . culture medium was replaced by bicarbonate - free , hepes - buffered mem ( icn biomedicals ) containing 0 . 5 % calf serum ( 0 . 1 ml / apical chamber ; 0 . 5 ml / basciateral chamber ), and the transwells were then incubated for 2 hours under a humidified air atmosphere at 37 ° c . it was verified that the switch to culture medium in itself did not affect the resistance of the mdck cells . additions were made to the chambers as indicated , and immediately 1 μci of [ u - 14 c ] sucrose ( 621 ci / mol ; amersham ) in 10 μl of culture medium was added to the apical chamber . the transwells were then shaken at setting 4 on a lucklam r100 rotary shaker ( denley instruments ltd ., west sussex . uk ), and aliquots of the basolateral chamber were removed at the indicated times for scintillation counting . cells were fixed in 3 pararormaldehyde made up in pbs containing 0 . 5 mm cacl 2 and 0 . 5 mm mgso 4 . after 15 minutes at room temperature , the fixed cells were washed and then permeabilized by incubation with 0 . 5 %. triton x - 100 in pbs for 10 minutes . after washing , the cells were incubated for 30 minutes in pbs containing 10 % calf serum plus 0 . 1 m lysine , ph 7 . 4 . incubation with primary antibody was in pbs containing 10 % calf serum for either 1 h at room temperature or overnight at 4 ° c . after washing , the cells were then incubated for 30 - 60 minutes at room temperature with a 1 : 1000 dilution of fitc - conjugated goat anti - mouse or anti - rat igg , as appropriate , in pbs containing 10 % calf serum . when required , rhodamine phalloidin ( 2 u / ml ; molecular probes , inc .) was included with the secondary antibody . after washing , the filters were mounted with citfluor ( citifluor products , canterbury , uk ) and examined using a nikon microphot - fxa fluorescence microscope fitted with 40 × and 60 × objectives . photographs were taken using kodak t - max film ( 400 asa ). confluent monolayers of cells on 24 min transwells were washed with ice - cold pbs ( containing 0 . 9 mm cacl 2 and 0 . 5 mm mgcl 2 ). after lysis in 0 . 5 ml of the appropriate buffer , the filters were gently scraped and the lysate was then centrifuged at 14 , 000 × g . the supernatant was precleared with 10 % ( w / v ) protein a - sepharose in lysis buffer for 15 minutes . appropriate primary antibodies were added to the lysate and the immune complex was isolated using rabbit secondary antibodies , as necessary , and protein a - sephrose . the beads were washed five times with lysis buffer , and bound protein was solubilized in sds - sample buffer ( laemmli , 1970 ) followed by heating at 100 ° c . for 5 minutes . the protein inhibitors used were : leupeptin 10 μg / ml ; α 2 - macroglobulin , 0 . 1 u / ml ; soybean trypsin inhibitor , 10 μg / ml ; pmsf , 1 mm . the cadherin / catenin complex was isolated at 4 ° c . using buffer comprising : 20 mm imidazole , ph 6 . 8 ; 100 mm kcl ; 10mm egta : 2 mm mgcl 2 ; 300 mm sucrose ; 0 . 2 % triton x - 100 ; 1 mm vanadate ; 1 mm naf ; 25 μm pao ; 0 . 02 % nan 3 ; protease inhibitors . denatured proteins were isolated by immunopreciptiation after lysing the cells in : 1 % sds ; hepes , ph 7 . 4 ; 25 mm naf ; 1 mm vandata ; 25 μm pao ; 2 mm edta . the lysates were heated for 5 minutes at 100 ° c . dna was shared using a 23 g needle and the sample was then diluted into 2 ml of ice - cold buffer comprising : 3 % triton x - 100 ; 0 . 1 m nacl ; hepes , ph 7 . 4 ; 4 . 25 mm naf ; 1 mm vanadate ; 25 μm pao ; 2 mm edta ; protease inhibitors . zo - 1 and associated proteins were isolated at 4 ° c . essentially as described by cumbiner et al . ( 1991 ) using buffer comprising : 1 % triton x - 100 ; 0 . 5 % sodium deoxycholae ; 0 . 2 % sds ; 0 . 15 m nacl ; 10 mm hepes , ph 7 . 4 ; 25 mm naf ; 25 μm pao ; 2 mm edta ; protease inhibitors . whole cell lysates were prepared by rapidly replacing the culture medium with sds sample buffer ( laemmli , 1970 ) followed by heating at 100 ° c . for 5 minutes . cell extracts or immunoprecipitates in sds sample buffer were resolved by sds - page ( laemmli , 1970 ). the slab gels were then equilbrated in buffer comprising : 48 mm tris , 39 mm glycine , 20 % methanol and 0 . 03 % sds . proteins were trasferring to nitrocellulose filters ( hybond ecl ; amersham ) which were ponceau s stained and then blocked in 5 % non - fat dried milk in pbs . after washing in pbs containing 0 . 05 % tween - 20 , the filters were probed with horseradish peroxidate - conjugated anti - phosphoryosine antibody ( rc20h ) or other unconjugated antibodies as described . after washing , unconjugated antibody was reacted with the appropriate horseradish peroxidase - conjugated secondary antibody . the filters were then extensively washed and immunoreactive bands were detected by enhanced chemiluminescence ( amersham ) following the manufacturer &# 39 ; s instructions . the anti - canine e - cadherin antibody rr1 developed by gumbiner and simons ( 1996 ) was provided by barry gumbiner ( memorial sloan - kettering cancer center , ny ) or obtained from the developmental studies hybridoma bank maintained by the department of pharmacology and molecular sciences , johns hopkins university school of medicine , baltimore , md . 21205 , and the department of biological sciences , university of iowa , iowa city , iowa 52242 , under contract n01 - hd - 2 - 3144 from the nichd . the anti - human e - cadherin antibody hecd - 1 ( shimoyama et al ., 1989 ) was from takara biomedicals ( shiga , japan ). anti - p120 and anti - focal adhesion kinase ( fak ) antibodies were from transduction laboratories ( lexington , ky .). the anti - p120 antibody 2b12 ( kanner et al ., 1990 ) was a gift from j . t . parsons ( university of virgina , charlottesville , va .). the peptide - directed antibodies against α - and β - catenin ( staddon et al ., j . cell sci . in press ) were kindly provided by kurt herrenknecht ( eisai research laboratories ltd ., university college london , london uk ). all secondary antibodies used for immunoprecipitation and immunocytochemistry were from jackson laboratories inc . ( west grove , pa .). hrp - conjugated secondary antibodies used for immunoblotting were from amersham ( buckinghamshire , uk ). the following cells were cultured at 37 ° c . in medium containing 100 u / ml penicillin and 100 μg / ml strepomycin ; caco - 2 ( epithelial cells derived from a human colonic tumour : 5 % co 2 , mem , 10 % fcs , 1 % non - essential amino - acids 1 μg / ml insulin ); ecv304 ( a cell line derived from human vein endothelial cells : 5 % co 2 , m199 , 10 % fcs ); llc - pk1 ( epithelial cells derived from porcine kidney : 5 % co 2 , m199 , 10 % fcs ); mdbk ( epithelial cells derived from bovine kidney : 5 % co 2 , mem10 % fcs ); strain i mdck cells ( epithelial cells derived from canine kidney : 5co 2 , mem , 10 % fcs ); rbe4 cells ( immortablized rat brain endothehal cells ( see duneu - trautmam et al ., 1993 ): 5 % co 2 , α - mem : ham &# 39 ; s f10 ( 1 : 1 ), 10 % fcs , 0 . 3 mg / ml geneticin , 1 ng / ml bfgf ); swiss 3t3 fibroblasts ( 10 % co 2 , dmem , 10 % fcs ). caco - 2 , evc304 , ccl - pk1 and mdbk cells were obtained form the european collection of animal cell cultures ( salisbury , uk ), mdck cells were provided by barry gumbiner , rbe4 cells were from pierre couraud ( universitéparis vii , paris , france ) and swiss 3t3 fibroblasts were enrique rozengutt ( imperial cancer research fund , london , uk ). human umbilical vein endothelial cells were from clonetics ( palo alto , calif .) and culture according the manufacturer &# 39 ; s instructions . primary cultures of bovine or porcine brain endothelial cells were grown as described by rubin et al . ( 1991 ). for experimental purposes , confluent cultures of caco - 2 , mdbk , mdck and brain endothelial cells were established on tissue culture - treated , polycarbonate transwell filters ( polycarbonate , 0 . 4 μm ; costar , cambridge , mass .). other cells were grown on tissue culture plastic . whole cell lysates from cultures maintained for 16 - 20 hours in 0 . 5 % serum were prepared by rapidly replacing the medium with hot laemmli sample buffer ( laemmli , 1970 ) supplemented with 5 mm edta , followed by heating at 100 ° c . for 5 minutes . proteins were resolved by slab - gel electrophoresis as described by laemmli , ( 1970 ). the gels were equalibrated in buffer containing 48 mm tris , 39 mm glycine , 0 . 03 % sds ( w / v ) and 20 % methanol ( v / v ), and then transferred to nitrocellulose filters ( hybond , ecl , amersham ). after ponceau s staining , the filters were blocked in 5 % ( w / v ) non - fat dried milk in pbs at 4 ° c . for 16 - 18 hours . filters were then incubated with primary antibody in pbs containing 0 . 05 % tween - 20 and 1 % bsa , followed by detection with appropriate hrp - conjugated secondary antibody and chemiluminescence ( ecl , amersham ). immunoprecipitations were performed at 4 ° c . cultures were rinsed with pbs and then lysed in either tx buffer ( 1 % ( v / v ) triton x - 100 , 25 mm hepes , 2 mm edta , 0 . 1 m nacl , 25 mm naf , 1 mm vanadate , 25 μm phenylarsine oxide , ph 7 . 6 ( adjusted with naoh ), 1 mm pmsf , 10 μg / ml soybean trypsin inhibitor , 0 . 1 u / ml α 2 - macroglobulin , 10 μg / ml leupeptin ) or tds buffer , which was identical to the tx buffer except that it was supplemented with 0 . 5 % ( w / v ) sodium deoxycholate and 0 . 2 % ( w / v ) sds . the cells were incubated with lysis buffer for 10 - 15 minutes and then scraped . the lysates were collected and centrifuged at 14 , 000 × g for 20 minutes . the supernatant was precleared with protein a sepharose ( pharmacia , uk ) for 1 - 2 hours and then incubated with primary antibody for 1 hour followed by a further 1 hour with protein a sepharose alone , in the case of rabbit antibodies , or together with rabbit anti - mouse antibodies for the mouse monoclonal antibodies . after five washes in lysis buffer , immune complexes were dissociated by addition of laemmli sample buffer followed by heating at 100 ° c . for 5 minutes . protein analysis was by sds - page and immunoblotting as described above . for [ 35 s ] methionine labelling , the cultures were washed twice in methionine - free mem supplemented with 0 . 5 % fcs . the cells were incubated for 16 - 18 hours in this medium containing 50 μc / ml [ 35 s ] methionine (& gt ; 1000 ci / mmol , amersham ). protein analysis was by sds - page , followed by fixation in 25 % methanol / 10 % acetic acid . labelled protein was detected either by direct autoradiography at room temperature or by fluorography at − 80 ° c . following impregnation of the gel with amplify ( amersham ). cells were fixed at room temperature for 15 minutes in 3 % paraform - aldehyde made up in pbs containing 0 . 5 mm cacl 2 and 0 . 5 mm mgso 4 . fixed cells were washed and then permeabilized by incubation with 0 . 5 % triton x - 100 in pbs for 10 minutes . after washing , the cells were incubated for 30 minutes in pbs containing 10 % calf serum and 0 . 1 m lysine , ph 7 . 4 . incubation with primary antibody was in pbs containing 10 % calf serum for 1 hour . after washing , the cells were then incubated for 30 - 60 minutes with a 1 : 100 dilution of fluorophore - conjugated anti - mouse or anti - rabbit igg , as appropriate , in pbs containing 10 % calf serum . after washing , the filters were mounted with citifluor ( citifluor products , canterbury , uk ) and examined using a nikon microphot - fxa fluroescence microscope fitted with 40 × and 60 × objectives . photographs were taken using kodak t - max film ( 400 asa ). for the preparation of cryosections , brain and skeletal muscle from co 2 - asphyxiated rats were removed and rapidly frozen in liquid nitrogen . tissue blocks were mounted in tissue tek ( r . lamb , london , uk ) and sections of 5 - 10 μm thickness were cut one a bright cryostat , air - dried and stored for up to 4 weeks at − 20 ° c . after thawing , the sections were fixed and permeabilized as described above . the sections were then washed , blocked with pbs containing 10 % calf serum for 15 minutes and incubated with primary antibody diluted in pbs containing 10 % calf serum for 2 hours . after washing , they were incubated with pbs containing 10 % calf serum with either 10 % goat serum or 10 % donkey serum , as appropriate of the host of the secondary antibody , for 15 minutes . they were then incubated with secondary antibody diluted in pbs and serum for 1 hour . sections were washed , mounted and examined as described above . pervanadate decreases the transcellular electrical resistance of mdcki cells . strain i mdck cells were grown on 6 . 5 mm , 0 . 4 μm pore , transwell ™ filters ( costar catalogue no . 3413 ). the cells were plated at 10 5 per filter and incubated for 4 - 7 days at 37 ° c . in a humidified 5 % co 2 incubator . under these conditions the strain i mdck cells gave transcellular electrical resistance values of 2000 - 5000 ω - cm 2 . in the experiment whose results are shown graphically in fig1 a , vanadate was added ( as the sodium salt ph7 ), to a final concentration of 0 . 01 mm (▪) 0 . 1 mm () or 10 mm (▴) transcellular electrical resistance ( ter ) was measured , with respect to control cells (□), using the millicell - ers ™ system from millipore ( uk ) limited at the times indicated . after 3 hours , h 2 o 2 was added to a final concentration of 2 mm ( arrow ). in the experiment whose results are shown graphically in fig1 b , vanadate and h 2 o 2 were preincubated prior to addition to the cells and excess h 2 o 2 was removed by catalase . the pervanadate was then added to the cells to give a final concentration of 0 . 01 mm (▪), 0 . 03 mm () or 0 . 1 mm (▴), and ter was determined with respect to control cells (□) and cells to which the ca 2 + chelator bapta was added (◯, 2 . 5 mm final concentration ). in the experiment whose results are shown in fig1 c , the cells were incubated with 0 . 1 mm (▪) or 1 . 0 , m vanadate (), and ter was determined with respect to control cells (□). in all cases , the resistance values have been expressed relative to the initial resistance . phenylarsine oxide decreases the transcellular electrical resistance of mdck i cells . stain i mdck cells were grown on transwell filters under the conditions described in example 1 . in the experiment whose results are shown graphically in fig2 a , phenylarsine oxide was added to a final concentration of 1 μm (▪), 3 μm (), 10 μm (▴), or 30 μm (♦) and the ter was expressed relative to the control cells (□) . in the experiment whose results are shown graphically in fig2 b phenylarsine oxide ( pao ) or bapta was added to a final concentration of 10 μm and 3 mm , respectively , to cells that had been preincubated for 10 minutes with 200 μm β - mercapoethanol ( βme ) or 100 μm 2 , 3 - dimercaptopropranol ( dmp ). ter was determined 30 minutes later . the values shown are the mean ± s . d . of values derived from triplicate transwell filters , except for the bapta measurements which were the average of two filters . pervanadate ( pv ) was added to a final concentration of 0 . 1 mm to cells that had been preincubated for 10 min in the absence or presence of 500 μm β - mercaptoethanol or 250 μm 2 , 3 - dimercaptopropanol . ter was determined 30 minutes later . the values shown are the means of determinations from two transwell filters . reversibility of the decrease in transcellular electrical resistance induced by the tyrosine phosophatase inhibitors . strain i mdck cells were grown as described in example 1 on transwell filters . in the experiments whose results are shown in fig3 a , the cells were treated in the absence () or presence of 100 μm pervanadate (▪,▴,) 2 , 3 - dimercaptopropanol ( 250 μm ) was added 10 min (▪) , 20 min (▴) or 30 min () after pervanadate . ter was determined at the times indicated . in the experiment whose results are shown in fig3 b , the cells were treated in the absence (□) or presence of 10 μm phenylarsine oxide (▪,, ▴). dimercaptopropanol 100 μm ) was added 15 min (▪), 30 min () or 45 min (▴) after phenylarsine oxide . ter was determined at the times indicated . the values shown ( means ± s . d . of triplicate transwell filters ) have been expressed relative to the initial ter . pervanadate and phenylarsine oxide decrease the transcellular electrical resistance of brain capillary endothelial cells . bovine brain capillary endothelial cells were grown on transwell filters and treated for two days in the absence ( fig4 b and 4 d ) or presence ( fig4 a and 4 c ) of cpt - camp / ro20 - 1724 , as described by rubin et al , j . cell biol . 115 1725 - 1735 ( 1991 ). the data in fig4 a / 4 b and fig4 c / 4 d were derived from two independent preparations of cells , giving different initial resistance values . in fig4 a / 4 b , the cells were incubated in the absence (□) or presence of 1 μm (▪), 3 μm () , 10 μm (▴), 30 μm (♦) or 100 μm (▾) pervanadate . ter was determined at the times indicated . in fig4 c / 4 d , the cells were treated in the absence (□) or presence of 0 . 3 μm (▪), 1 μm (), 3 μm (▴), 10 μm (♦), 30 μm (▾) phenylarsine oxide . ter was determined at the times indicated . pao and pervanadate increase the paracellular flux of sucrose in mdck cells . sucrose flux was determined as described under materials and methods . flux ( mean ± s . e ., n = 1 . 6 ) in repsonse to 10 μm pao , 100 μm pervanadate ( pv ) or 4 mm bapta was determined with respect to control cells ( cont .) ( see fig4 . 1 ). pervanadate increases the tyrosine phosphorylation of many proteins in mdck strain i cells and bbecs : comparison with the discrete effects of phenylarsine oxide . mdck strain i cells ( fig5 a ) or cyclic amp treated bbecs ( fig5 b ) were treated in the absence ( cont .) or presence of 100 μm pervanadate ( pv ) for 30 min . in fig5 c , mdck strain in cells were incubated with 10 μm phenylarsine oxide for the indicated times . in fig5 d , cyclic amp treated bbecs were treated with the indicated concentrations of phenylarsine oxide for 60 min . in all cases , the cells were lysed with sds - containing buffer . solubilised proteins were separated by sds - page and transferred to nitrocellulose . tyrosine phosdhorylation was detected using an anti - phosyhotyrosine antibody conjugated to horseradish peroxidase followed by enhanced chemiluminescence . it should be noted that the exposure times ( seconds ) to obtain the results shown in fig5 a and 5 b were considerably shorter than those ( minutes ) to obtain the results in fig5 c and 5 d . with respect to the bbecs , similar results were obtained if the cells were treated with or without cyclic amp . pao increases the tyrosine phosphorylation of proteins at intercellular junctions in mdck cells comparison with the effect of pervanadate . mdck cells were treated in the absence ( a , f ) or presence of either 100 μm pervanadate for 15 ( b , g ) or 30 minutes ( c , h ) or with 10 μm pao for 15 ( d , i ) or 30 minutes ( e , j ). the cells were stained with an anti - phosphotyrosine antibody ( a - f ) or an anti - e - cadherin antibody ( g - j ), as described under materials and methods , exposure times were constant for panels a - f . bar , 10 μm ( see fig5 . 1 ). pao increases the tyrosine phosphorylation of proteins at intracellular junctions in brain endothelial cells : comparison with the effect of pervanadate . filter - grown brain endothelial cells were treated with cpt - camp / ro20 - 1724 for 2 days as described by rubin et al . ( 1991 ). the cells were then incubated in the absence ( a , e ) or presence of either 10 μm pao for 5 ( b ), 10 ( c ) or 30 minutes ( d ) or 100 μm pervanadate for 5 ( f ), 10 ( g ) or 30 minutes ( h ). the cells were then fixed and stained with anti - phosphotyrosine antibody as described under materials and methods . exposure times were constant for panels a - d and constant , but less , for panels e — h . bar , 20 μm ( see fig5 . 2 ). pao stimulates the tyrosine phosphorylation of components of the e - cadherin / catenin complex in mdck cells . fig6 a : filter - grown cells were untreated ( cont .) or incubated for 30 minutes with 10 μm pao or for 15 minutes with 100 μm pervanadate ( pv ). the cells were lysed in buffer containing triton x - 100 and the e - cadherin / catenin complex was immunoprecipitated using anti - e - cadherin antibody . proteins were resolved by sds - page and tyrosine phosphorylation was detected by immunoblotting with anti - phosphotyrosine antibody . clearly , pao stimulates the tyrosine phosphorylation of a catenin of approximate molecular mass 100 kda . pervanadate stimulates the tyrosine phosphorylation of several catenins as well as e - cadherin . fig6 b , c , d , e : mdck cells were incubated with either 10 μm pao or 100 μm pervanadate ( pv ) for the indicated items . the cells were lysed into denaturing buffer ( containing sds ) to dissociated protein complexes and individual tyrosine phosphoproteins were immunoprecipitated using phosphotyrosine antibody . the immunoprecipitates were analyzed for either β - catenin ( arrow , fig6 b ) or α - catenin ( arrow , fig6 c ) content by immunoblotting . identical lysates were immunoprecipitated using peptide - directed α - catenin antibody and blotted with the same antibody ( fig6 d ), to confirm the success of the immunoprecipitation , or phosphotyrosine antibody ( fig6 e ). arrows indicate the migration of α - catenin . the exposure times to obtain the negative results shown in fig6 c and 6e were similar to those used to obtain the result in fig6 a . clearly , the β - catenin content of the phosphotyrosine immunoprecipitates from pao or pervanadate treated cells is increased ( fig6 b ), in contrast , the α - catenin content is not ( fig6 c ). similarly , tyrosine phosphorylation ( fig6 e ) of α - catenin immunoprecipitates ( fig6 d ) could not be detected . fig6 f and 6 g : mdck cells were untreated ( cont . ), incubated with 10 μm pao for either 15 minutes and then 100 μm 2 , 3 - dimercaptopropanol for a further 45 minutes ( pao 15 ′/ dmp 45 ′). the e - cadherin / catenin immunoprecipitate was analysed by immunoblotting for phosphotyrosine ( fig6 f ) or α - catenin content ( fig6 g ). the arrow in fig6 f indicates the migration of the tyrosine phosphorylated band seen in fig6 a . the arrow in fig6 g indicates the migration of α - catenin . the tyrosine phosphorylation of the catenin is reversed by the subsequent addition of 2 , 3 - dimercaptopropanol ( fig6 f ). the constant content of α - catenin in the immunoprecipitates ( fig6 g ) indicates quantitatively successful immunoprecipitation in all cases . pao stimulates the tyrosine phosphorylation of tight junction associated proteins in mdck cells . mdck cells were treated with 10 μm pao or 100 μm pervanadate for the indicated times . the cells were lysed in buffer containing triton x - 100 , sds and sodium deoxycholate . zo - 1 and associated proteins were immunoprecipitated using zo - 1 antibody and analyzed for phosphotyrosine content by immunoblotting . clearly , pao treatment resulted in the tyrosine phosphorylation of zo - 1 ( 220 kda ). pervanadate also stimulated tyrosine phosphorylation of zo - 1 , as well as zo - 2 ( 160 kda ) ( see fig7 ). herbimycin a attenuates the pao - induced decrease in transcellular electrical resistance in mdck cells . in two independent experiments , cultures were incubated in the absence or presence of the tyrosine kinase inhibitor herbimycin a (◯, control ; , 2 . 5 μm herbimycin a : □, control ; ▪, 10 μm herbimycin a ) for 16 - 18 hours after which a resistance measurement was taken . the addition of herbimycin a alone did not affect resistance . pao wash then added to give the final concentrations indicated and after a further 30 minutes incubation the effects on resistance were determined . the resistance values have been expressed relative to the initial values ( see fig8 ). herbimycin a attenuates the pao - induced increase in protein tyrosine phosphorylation in mdck cells . the cultures that have been used to generate the data testing the effects of 10 μm herbimycin a in example 8 were lysed in sds sample buffer and separated by sds - page . protein tyrosine phosphorylation was detected by immunoblotting using anti - phosphorylation was detected by immunoblotting using anti - phosphotyrosine antibody . ha , herbimycin a ( see fig9 ). middle cerebral artery occlusion ( a stroke model ) elevates the levels of tyrosine phosphorylation in cells associated with small blood vessels near the area damaged by the occlusion . the left middle cerebral artery of adult male sprague - dawley rats was exposed by craniotomy and occluded by cauterization . this gives a useful stroke model . twenty four hours after the operation . evan &# 39 ; s blue ( 4 % in pbs . 6 μl per gram body weight ) was injected intravenously . after thirty minutes , the animal was anaesthetised , the blood flushed out with pbs , the brain removed and snap frozen in powdered dry ice . sections of unfixed tissue were made using a bright otf cyrostat . sections ( 24 μm ) were air - dried on gelatin - coated glass slides , fixed for 10 minutes in methanol at − 20 ° c ., washed in pbs ( phosphate - buffered saline ) and mounted in citifluor . sections were air - dried and permeablised with 95 % ethanol at − 95 ° c . for 30 minutes followed by acetone at room temperature for 1 minute . all of the antibody incubations and washes used pbs containing 5 × 10 − 4 m sodium vanadate . sections were washed with pbs , blocked with 1 % bsa ( bovine serum albumin ) for 15 minutes and incubated overnight at 4 ° c . with either 10 μg / ml anti - phosphotyrosine antibody ( 4g10 ) or control antibody ( 1 : 100 ) together with anti - collagen type iv ( 1 : 40 , 000 ). sections were then blocked for 15 minutes in pbs / vanadate containing 1 % bsa and 0 . 1 % triton - x100 , incubated with fitc - conjugated anti - mouse igg2b ( 1 : 100 ) and texas red - conjugated anti - rabbit ig ( 1 : 100 ) for 1 hour at room temperature and mounted in citifluor . for some sections , anti - rat albumin antibody ( 1 : 200 ) replaced the 4g10 antibody , and in this case fitc - conjugated anti - sheep igg ( 1 : 100 ) was used instead of the anti - mouse igg2b . anti - phosphotyrosine antibody 4g10 was from upstate biotechnology inc . ( tcs biologicals , buckingham , uk ), sheep anti - rat albumin from biogenesis ( bournemouth , uk ), control mouse igg2b from zymed ( cambridge bioscience , cambridge , uk ), collagen type - iv from biogenesis , fitc - conjugated anti - mouse igg2b from nordic immunological laboratories ltd . ( maidenhead , uk ) and texas red - conjugated anti - rabbit ig and fitc - conjugated anti - sheep igg were from jackson immunoresearch laboratories inc . ( stratech , luton , uk ). sections were viewed on a nikon microphot fxa microscope using phase or nomarski optics to determine morphology , and with epifluoresence to visualize the fluorophores . excitation of texas red and evan &# 39 ; s blue was at 510 - 560 nm and fitc at 495 nm . 4g10 antibody was incubated for 15 minutes with 3 - 40 mm phosphotyrosine ( sigma chemical company ltd ., poole , uk ) before staining the section as described above . fig1 a , c and e : collagen type iv , collagen type iv is in the basement membrane around endothelial and smooth muscle cells and the antibody outlines blood vessels . fig1 b , d and f : phosphotyrosine label of the same sections shown in fig1 a , c and e . phosphotyrosine is not detectable in the microvessels in the normal tissue of the right cortex of an operated rat ( fig1 b ), but is increased in vessels near the damaged area in the cortex on the left side of the same brain ( fig1 d ). artificial elevation of the levels of phosphotyrosine using pervanadate induces detectable levels of phosphotyrosine in both large and small vessels ( fig1 f ( note that fig1 f is inverted )). characterization of anti - p120 and 2b12 immunoreactivity . various cell lines and a primary culture of bovine brain endothelial cells ( brain ec ) were lysed in sds sample buffer and analyzed by sds - page followed by immunoblotting with anti - p120 antibody ( panel a ) or the 2b12 antibody ( panel b ). the exposure times were 1 minute for panel a ( apart from that for the mdbk cells which was 10 seconds ) and 15 minutes for panel b . the migration of p120 (∘) and p100 () has been indicated ( see fig1 ). p120 protein is recognized by the anti - p120 antibody and cross - reacts with the 2b12 antibody , whereas p100 is recognized only by anti - p120 . mdbk cells were lysed in tds buffer . immunoprecipitations were performed using the anti - p120 antibody or 2b12 followed by cross - blotting ( panel a ). mdbk cells were chosen because p120 and p100 are well separated by sds - page and react with both antibodies . in panel b , mdbk cells were labelled with [ 35 s ] methionine , lysed in tds buffer and then immunoprecipitated using anti - p120 or 2b12 . proteins were separated by sds - page and detected by autoradiography . clearly , the broad bands ( panel 12 a and see fig1 a ) corresponding to p120 and p100 ass detected by immunoblotting are resolved as multiple bands . the same bands as seen in the 2b12 immunoprecipitate (∘) are seen in the anti - p120 immunoprecipitates . in the anti - p120 immunoprecipitates , additional bands () corresponding to p100 are also observed ( see fig1 ). comparison of anti - p120 and anti - e - cadherin immunoprecipitates from mdck and caco - 2 cells . panel a : mdck cells were labelled with [ 35 s ] methoionine and then lysed in either tx buffer or tds buffer . immunoprecipitations were performed using anti - p120 or anti - e - cadherin ( rr1 ). proteins were separated by sds - page followed by fluorography . bands corresponding to e - cadherin ( e ), α -( α ) and β - catenin ( β ) which are seen strongly in the rr1 immunoprecipitates have been indicated . the major anti - p120 reactive band migrates at approximately the position of that of p100 ( see fig1 a ). 2b12 does not appear to react with canine protein ( see fig1 b ) making positive identification of p120 difficult , panel b : caco - 2 cells were labelled with [ 35 s ] methionine and then lysed in tx buffer . immunoprecipitations were performed using anti - e - cadherin ( hecd - 1 ), anti - β - catenin or anti - p120 antibodies . proteins were separated by sds - page followed by fluorography . in all cases , four major comigrating bands were immunoprecipitated , corresponding in order of increasing mobility to e - cadherin , α -, β - and γ - catenin . ( see fig1 ). detection of anti - p120 reactive material in anti - e - cadherin immunoprecipitates and e - cadherin in the anti - p120 immunoprecipitates . mdck cells were lysed in tx buffer . immunoprecipitations were performed with either antibody to e - cadherin ( rr1 ), the anti - p120 antibody or anti - focal adhesion kinase ( anti - fak ). a sham immunoprecipitation was performed in the absence of primary antibody (−). following separation by sds - page , parallel blots were probed using : rr1 ( arrowhead , e - cadherin ); anti - β - catenin ( arrowhead , β - catenin ); anti - p120 ( arrowhead , anti - p120 reactivity ); anti - fak ( arrowhead , fak ). clearly , rr1 and the anti - p120 antibody immunoprecipitate e - cadherin and β - catenin . rr1 could also immunoprecipitate anti - p120 immunoreactive material , but to a lesser extent than that immunoprecipitated by the anti - p120 antibody . anti - fak could only immunoprecipitate fak , and the sham immunoprecipitation did not result in any detectable e - cadherin , β - catenin or anti - p120 reactive material ( see fig1 ). comparison of anti - β - catenin , anti - p120 and 2b12 immunoprecipitates from primary cultures of bovine brain endothelial cells . cells were labelled with [ 35 s ] methionine and then lysed in tx buffer or tds buffer . immunoprecipitations were performed using anti - β - catenin , anti - p120 and 2b12 . proteins were separated by sds - page followed by fluorography . in the β - catenin immunoprecipitation from tx lysed cells , bands corresponding to a cadherin ( c ), α - catenin ( α ) and β - catenin ( β ) have been indicated . under tds conditions , a band (∘) corresponding to p120 is seen in the anti - p120 and 2b12 immunoprecipitates and a further band () is only seen in the anti - p120 immunoprecipitate , corresponding to p100 ( see fig1 ). detection of p120 / p100 in β - catenin immunoprecipitates and β - catenin in anti - p120 immunoprecipitates from human umbilical vein endothelial cells ( panel a ) and ecv304 cells ( panel b ). cells were lysed in either tx or tds buffer . lysates were immunoprecipitated using anti - β - catenin or the anti - p120 antibody , followed by analysis by sds - page and immunoblotting . the migration of β - catenin ( arrowheads ), p100 () and p120 (∘) have been indicated . note the absence of anti - p120 reactive material in the β - catenin immunoprecipitates obtained using tds buffer . even though β - catenin and p100 , as defined by its reactivity with anti - p120 antibody , migrate very closely , they are clearly distinct proteins ( see fig1 ). localization of anti - p120 reactivity and β - catenin in mdck cells , and brain endothelial cells . mdck cells ( panel a , b ) and porcine brain endothelial cells ( panel c , d ) were co - labelled with anti - p120 antibody ( panel a , c ) and anti - β - catenin antibody ( panel b , d ). secondary antibodies were fluorescein - conjugated anti - mouse and rhodamine - conjugated anti - rabbit . in this instance , it was verified that each secondary antibody was absolutely specific for its designated species of primary antibody . bar : 20 μm ( see fig1 ). distribution of anti - p120 immunoreactivity in brain and skeletal muscle tissue of the rat . brain tissue was co - labelled with the anti - p120 antibody ( a , c ) and anti - α - catenin ( b , d ). anti - p120 immunoreactivity and α - catenin co - locaiise at intercellular junction of choroid plexus epithelium ( arrows , a and b ) and ventrical ependymal cells ( arrowheads , a and b ). both antigens also co - locaiise at interendothelial junctions of blood vessels of macrovascular origin ( arrows in c and d ). microvascular profiles in brain sections were identified by labelling with anti - collagen iv antibody ( f ); co - labelling with the anti - p120 antibody ( e ), revealed the presence of anitgen at interendothelial junctions ( e , arrows ). in these microvessels , α - catenin co - localised with anti - p120 immunoreactivity ( not shown ). in muscle tissue which had been cut perpendicular to the orientation of the muscle fibres , anti - p120 immunoreactivity is limited to areas between muscle fibres where blood vessels are located ( g , arrows ). higher magnification reveals a punctate staining pattern ( h , arrows ) which is likely to reflect anti - p120 immunoreactivity at interendothelial junctions . in panels a , c and e , bp depicts the brain parenchyma ; in panel a , v the ventrical lumen and cp the choroid plexus ; in panel g , m refers to muscle tissue . bars : ( a , g ), 100 μm ; ( c , e , h ), 25 μm ( see fig1 ). sequence analysis of peptides derived by lysc proteolysis of p100 from caco - 2 cells . caco - 2 cells ( 20 × confluent 9 cm dishes ) were lysed in tx buffer to minimize nuclear lysis . insoluble protein was removed by centrifugation . deoxycholate and sds were added to the supernatant to give ( w / v ) 0 . 5 % and 0 . 2 % final concentration , respectively . addition of these detergents results in dissociation of p100 and p120 from the cadherin / catenin complex . p120 and p100 were immunoprecipitated from the lysate using the anti - p120 antibody ( which also recognizes p100 ) from transduction laboratories , rabbit anti - mouse igg and protein a sepharose . the immune complex was washed five times and then dissociated by the addition of laemmli sample buffer followed by heating at 100 ° c . for 5 minutes . proteins were precipitated by addition of four volumes of ethanol and incubation at − 20 ° c . for 16 hours . the precipitate was resolved by sds - page ( 6 % acrylamide ) and proteins were visualized by coomassie blue . protein corresponding to p100 was excised from the gel and digested with lysc . peptides were separated by hplc and sequenced ( seq id no : f9 ). mouse p120 sequence was described by reynolds et al ., 1992 . clearly , human p100 is closely related to mouse p120 ( seq id no : 519 ). the following reference list gives details of the articles referred to herein . anderson j . m ., balda , m . s ., and fanning , a . s . 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