- Email: [email protected]
Plasmodium berghei: Induction of Aminopeptidase in Malaria-Resistant Strain of Anopheles gambiae
Experimental Parasitology 93, 101–104 (1999) Article ID expr.1999.4432, available online at http://www.idealibrary.com on
RESEARCH BRIEF Plasmodium berghei: Induction of Aminopeptidase in Malaria-Resistant Strain of Anopheles gambiae
Ahron Rosenfeld and Jerome P. Vanderberg1 Department of Medical and Molecular Parasitology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, U.S.A.
Rosenfeld, A., and Vanderberg, J. P. 1999. Plasmodium berghei: Induction of aminopeptidase in malaria-resistant strain of Anopheles gambiae. Experimental Parasitology 93, 101–104. q 1999 Academic Press Index Descriptors and Abbreviations: aminopeptidase; Anopheles gambiae; Plasmodium falciparum; Plasmodium berghei; amastatin; R, refractory; S, susceptible; BSA, bovine serum albumin.
specific R mosquito protein in response to infection with a malaria parasite, we followed up this observation with an attempt to define this phenomenon. Mosquitoes used were an An. gambiae strain (G3), which had been used as the original starting point for selection of the original R (L35) and S (4arr) strains (Collins et al. 1986). We used recently rederived colonies of these strains (Zheng et al. 1997) for our studies. We prepared mosquito midgut extracts and collected hemolymph as previously described (Rosenfeld and Vanderberg 1998) and then performed Native electrophoresis using the PhastSystem (Pharmacia). After electrophoresis, gels were tested for the ability of electrophoresed proteases to hydrolyze the aminopeptidase substrate L-leucine p-nitroanilide (Sigma). Strips of nitrocellulose soaked in 10 mg/2.5 ml substrate solution for 15 min were overlaid on electrophoresed gels. The gels were then incubated at 378C just until the yellow p-nitroaniline products of digestion appeared on the nitrocellulose membranes, which were then diazotized (Ohlsson et al., 1986) for better visualization. Quantification of band density was by analysis of scanned zymograms using the program, ImageQuant Version 3.3. Each of our studies was done in at least three independent experiments involving separate cohorts of mosquitoes; for each of these experiments, we performed at least two separate gel runs. Hemolymph components separated by Native–PAGE hydrolyzed the leucine aminopeptidase substrate to produce eight bands, while midgut material produced six bands (Fig. 1). These bands were barely detectable in sugar-fed mosquitoes at the levels of midgut extract applied (0.4 mg protein/lane) but became more apparent at higher concentrations of sugar-fed midgut extract or when midguts were dissected 24 h after a bloodmeal (Fig. 1). The most striking result was that within 24 h after a P. bergheiinfected bloodmeal taken by R mosquitoes, there was a fivefold increase in substrate digestion by a single band (designated AM2; arrowhead in
A long-term approach for vector-based control of malaria involves identification of mosquito factors concerned in mosquito rejection of malaria parasites, identification of gene(s) encoding proteins responsible for this, and then replacing Anopheles vector populations with refractory mosquitoes possessing the gene(s) (Crampton 1994). An important step toward this goal was the selection of An. gambiae strains either susceptible (S) or refractory (R) to several malaria species, including Plasmodium falciparum and P. berghei (Collins et al. 1986). One would expect to find significant molecular differences between S and R mosquitoes that are related to the differential ability of these mosquito strains to specifically recognize parasites as “nonself” invaders or to the differential ability of these mosquitoes to destroy these parasites. However, no such functional differences between S and R mosquitoes have yet been established. Accordingly, we initiated a survey using PAGE followed by zymography to search for differences in isozyme patterns between S and R An. gambiae. During this analysis, we observed that R mosquitoes exhibited a marked upregulation of one of their midgut aminopeptidase isozymes following a bloodmeal containing infective P. berghei gametocytes. As this appears to be the first described upregulation of a 1
To whom correspondence should be addressed. Fax: (212) 2638116. E-mail: [email protected].
0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
101
102
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103
AMINOPEPTIDASE INDUCTION IN An. gambiae BY P. berghei
Fig. 1) compared to what was observed in mosquitoes fed on noninfected hamsters or hamsters infected with an agametocytic strain of P. berghei (Anka 2.33, obtained from Dr. Robert Sinden of Imperial College, London). Finally, a band electrophoretically indistinguishable from AM2 was detectable in S mosquitoes after either an infective or a noninfective bloodmeal (Fig. 1). There was no evidence of upregulation associated with Plasmodium infection of S mosquitoes. To test the effects of protease inhibitors on the bands, zymography was done as described but with the addition of one of several inhibitors to the washing buffer and the chromagenic substrate. No significant inhibition of the aminopeptidase isozymes was detected after exposure to serine protease inhibitors (antipain–dihydrochloride [74 mM] or aprotinin [0.3 mM], nor to the cystine protease inhibitor E-64 [28 mM]). All bands were inhibited by the Zn21 chelator, 1,10-phenanthroline [10 mM], and by the aminopeptidase inhibitor, bestatin [130 mM]. Thus, all of the isobands met the criteria for being authentic aminopeptidases on the basis of their specificity to aminopeptidase substrates rather than substrates of other proteolytic enzymes (data not shown), their susceptibility to specific inhibitors of aminopeptidase activity and to a Zn21 chelator, and their insensitivity to inhibitors of serine proteases and cysteine proteases. Of particular interest was our observation of a differentially high inhibition of AM2 and the band immediately above it, after exposure to amastatin [10 mM], a specific inhibitor of some aminopeptidases. Our findings with the “immune” aminopeptidase isoband appears to be the first described upregulation of a specific An. gambiae protein in response to infection with a malaria parasite; furthermore, this upregulation was restricted to R mosquitoes. Recent studies have demonstrated upregulation of immune marker RNA’s specifically after a malaria-infected bloodmeal taken by An. gambiae (Dimopoulos et al. 1997; Richman et al. 1997). These studies could not, however, detect any differences between R and S mosquitoes. A potential serine protease candidate for involvement in host defense has been reported from An. gambiae (Han et al. 1997). The gene for this protease did not show increased transcription after a bloodmeal, suggesting its lack of involvement in bloodmeal digestion; moreover, there was greater expression of this gene in R than in S mosquitoes. Aminopeptidases have been shown to have a wide range of functions in both wound healing and host defense against invasive microorganisms. Elevation of aminopeptidase activity has been reported in human burn wound exudates (Prager et al. 1994), in tomato plants infected with Pseudomonas syringae (Pautot et al. 1993), and in strains of An. stephensi genetically selected for resistance to P. falciparum (Feldmann et al. 1990). Aminopeptidase activity is also upregulated in the serum of
Biomphalaria glabrata snails parasitized by the trematode Echinostoma lindoense, and it was suggested that the enzyme may act to uncover cryptic sites on the invader (Cheng et al. 1978). Thus, the striking elevation of AM2 in R mosquitoes 24 h after a P. berghei-infected bloodmeal could conceivably be related to host defense against the malaria parasite. However, the specific role, if any, of this putative “immune” aminopeptidase in a response against the malaria parasite remains to be established. When the aminopeptidase inhibitor bestatin was supplied to tomato plants, Schaller et al. (1995) observed induction of several defenseassociated genes, including the gene for leucine aminopeptidase. They suggested that bestatin may be exerting its effects close to the level of transcriptional control of these defense genes. Using a parallel approach, we fed a related synthetic inhibitor of aminopeptidase (amastatin at 1 mg/ml) within a noninfected hamster bloodmeal presented to R mosquitoes through a parafilm membrane and observed an upregulation of AM2 comparable to that observed following an infected bloodmeal (data not shown), thus suggesting an inhibitor-induced mechanism similar to that described by Schaller et al. (1995). For further studies with AM2, our ability to strikingly inhibit its activity with amastatin presents a powerful tool to investigate the in vivo function of this enzyme and to purify it by affinity chromatography using amastatin attached to agarose beads. (This investigation received financial assistance from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and through N.I.H. National Research Service Award 5T32AI07180-15 0071 from which Ahron Rosenfeld received stipend support as a pre-doctoral fellow.)
REFERENCES
Cheng, T. C., Lie, K. J., Heyneman, D., and Richards C. S. 1978. Elevation of aminopeptidase activity in Biomphalaria glabrata (Mollusca) parasitized by Echinostoma lindoense (Trematoda). Journal of Invertebrate Pathology 31, 57–62. Collins, F. H., Sakai, R. K., Vernick, K. D., Paskewitz, S., Seeley, D. C., Miller, L. H., Collins, W. E., Campbell, C. C., and Gwadz, R. W. 1986. Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 234, 607–610.
FIG. 1. The 8–25% gradient gel Native–PAGE enzymogram of Anopheles gambiae L35 (a) and “wild-type” G3 (b) midgut extract and hemolymph from mosquitoes that had been sugar fed (SF) or received a normal bloodmeal (NBM) or a Plasmodium berghei-infected bloodmeal (IBM) (post-24-h bloodmeal collection). For midgut samples, a total of 0.15 mosquito equivalents (0.4 mg of midgut protein) was loaded per lane. For hemolymph samples, 0.2 mg of hemolymph protein was loaded per lane. Gels were then run for 312 accumulated volt-h at 158C. Substrate used was L-leucine p-nitroanilide for the determination of aminopeptidase-like activity. Arrowhead denotes a band found to be upregulated after an infected bloodmeal in malaria-resistant L3-5 mosquitoes. The midgut aminopeptidase activity detected was determined to be of endogenous origin since controls such as uninfected hamster blood, infected hamster blood, mosquito salivary gland extract, and Malpighian tubule extract failed to yield similar bands when these control materials were applied to the gel in amounts equivalent to the amounts of midgut protein applied per lane. The far-left lane shows molecular mass standards (M): (thyroglobulin [669 kDa], ferritin [440 kDa], catalase [232 kDa], lactate dehydrogenase [140 kDa], and BSA [67 kDa]; Pharmacia). Because migration of nonreduced proteins during Native–PAGE depends upon charge density as well as molecular size of the protein (Rothe and Maurer 1986), the markers cannot be used directly to assess molecular masses of mosquito proteins but rather only as indicators of the relative location of electrophoretically separated proteases.
104 Crampton, J. M. 1994. Approaches to vector control: New and trusted. 3. Prospects for genetic manipulation of insect vectors. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 141–143. Dimopoulos, G., Richman, A., Mu¨ller, H. M., and Kafatos, F. C. 1997. Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites.Proceedings of the National Academy of Science USA 94, 11508–11513. Feldmann, A. M., Billingsley, P. F., and Savelkoul, E. 1990. Bloodmeal digestion by strains of Anopheles stephensi of differing susceptibility to Plasmodium falciparum. Parasitology 101, 193–200. Han, Y. S., Salazar, C. E., Reese-Stardy, S. R., Cornel, A., Gorman, M. J., Collins, F. H., and Paskewitz, S. M. 1997. Cloning and characterization of a serine protease from the human malaria vector, Anopheles gambiae. Insect Molecular Biology 6, 385–395. Ohlsson, B. G., Westro¨m, B. R., and Karlsson, B. W. 1986. Enzymoblotting: A method for localizing proteinases and their zymogens using para-nitroanilide substrates after agarose gel electrophoresis and transfer to nitrocellulose. Analytical Biochemistry 152, 239–244. Pautot, V., Holzer, F. M., Reisch, B., and Walling, L. L. 1993. Leucine aminopeptidase: An inducible component of the defense response in Lycopersicon esculentum (tomato). Proceedings of the National Academy of Science USA 90, 9906–9910. Phastsystem Owners Manual. 1986. Laboratory Separation Division, Pharmacia-LKB, Uppsala, Sweden.
ROSENFELD AND VANDERBERG
Prager, M. D., Sabeh, F., Baxter, C. R., Atiles, L., and Hartline, B. 1994. Dipeptidyl peptidase IV and aminopeptidase in burn wound exudates: implications for wound healing. Journal of Trauma 36, 629–633. Richman A., Dimopoulos, G., Seeley, D., and Kafatos, F. C. 1997. Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes. EMBO Journal 16, 101–106. Rosenfeld A., and Vanderberg J. P. 1998. Identification of electrophoretically-separated proteases from midgut and hemolymph of adult Anopheles stephensi mosquitoes. Journal of Parasitology 84, 361–365. Rothe, G. M., and Maurer, W. D. 1986. One dimensional PAA-gel electrophoretic techniques to separate functional and denatured proteins. In “Gel Electrophoresis of Proteins” (M. J. Dunn, Ed.), pp. 37–140. IOP Publishing Ltd., Bristol, U.K. Schaller, A., Bergey, D. R., and Ryan, C. A. 1995. Induction of wound response gene in tomato leaves by Bestatin, an inhibitor of aminopeptidases. The Plant Cell 7, 1893–1898. Zheng L., Cornel, A. J., Wang, R., Erfle, H., Voss, H., Ansorge, W., Kafatos, F. C., and Collins, F. H. 1997. Quantitative trait loci for refractoriness of Anopheles gambiae to Plasmodium cynomolgi B. Science 276, 425–428. Received 4 November 1998; accepted with revision 9 June 1999
RESEARCH BRIEF Plasmodium berghei: Induction of Aminopeptidase in Malaria-Resistant Strain of Anopheles gambiae
Ahron Rosenfeld and Jerome P. Vanderberg1 Department of Medical and Molecular Parasitology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, U.S.A.
Rosenfeld, A., and Vanderberg, J. P. 1999. Plasmodium berghei: Induction of aminopeptidase in malaria-resistant strain of Anopheles gambiae. Experimental Parasitology 93, 101–104. q 1999 Academic Press Index Descriptors and Abbreviations: aminopeptidase; Anopheles gambiae; Plasmodium falciparum; Plasmodium berghei; amastatin; R, refractory; S, susceptible; BSA, bovine serum albumin.
specific R mosquito protein in response to infection with a malaria parasite, we followed up this observation with an attempt to define this phenomenon. Mosquitoes used were an An. gambiae strain (G3), which had been used as the original starting point for selection of the original R (L35) and S (4arr) strains (Collins et al. 1986). We used recently rederived colonies of these strains (Zheng et al. 1997) for our studies. We prepared mosquito midgut extracts and collected hemolymph as previously described (Rosenfeld and Vanderberg 1998) and then performed Native electrophoresis using the PhastSystem (Pharmacia). After electrophoresis, gels were tested for the ability of electrophoresed proteases to hydrolyze the aminopeptidase substrate L-leucine p-nitroanilide (Sigma). Strips of nitrocellulose soaked in 10 mg/2.5 ml substrate solution for 15 min were overlaid on electrophoresed gels. The gels were then incubated at 378C just until the yellow p-nitroaniline products of digestion appeared on the nitrocellulose membranes, which were then diazotized (Ohlsson et al., 1986) for better visualization. Quantification of band density was by analysis of scanned zymograms using the program, ImageQuant Version 3.3. Each of our studies was done in at least three independent experiments involving separate cohorts of mosquitoes; for each of these experiments, we performed at least two separate gel runs. Hemolymph components separated by Native–PAGE hydrolyzed the leucine aminopeptidase substrate to produce eight bands, while midgut material produced six bands (Fig. 1). These bands were barely detectable in sugar-fed mosquitoes at the levels of midgut extract applied (0.4 mg protein/lane) but became more apparent at higher concentrations of sugar-fed midgut extract or when midguts were dissected 24 h after a bloodmeal (Fig. 1). The most striking result was that within 24 h after a P. bergheiinfected bloodmeal taken by R mosquitoes, there was a fivefold increase in substrate digestion by a single band (designated AM2; arrowhead in
A long-term approach for vector-based control of malaria involves identification of mosquito factors concerned in mosquito rejection of malaria parasites, identification of gene(s) encoding proteins responsible for this, and then replacing Anopheles vector populations with refractory mosquitoes possessing the gene(s) (Crampton 1994). An important step toward this goal was the selection of An. gambiae strains either susceptible (S) or refractory (R) to several malaria species, including Plasmodium falciparum and P. berghei (Collins et al. 1986). One would expect to find significant molecular differences between S and R mosquitoes that are related to the differential ability of these mosquito strains to specifically recognize parasites as “nonself” invaders or to the differential ability of these mosquitoes to destroy these parasites. However, no such functional differences between S and R mosquitoes have yet been established. Accordingly, we initiated a survey using PAGE followed by zymography to search for differences in isozyme patterns between S and R An. gambiae. During this analysis, we observed that R mosquitoes exhibited a marked upregulation of one of their midgut aminopeptidase isozymes following a bloodmeal containing infective P. berghei gametocytes. As this appears to be the first described upregulation of a 1
To whom correspondence should be addressed. Fax: (212) 2638116. E-mail: [email protected].
0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
101
102
ROSENFELD AND VANDERBERG
103
AMINOPEPTIDASE INDUCTION IN An. gambiae BY P. berghei
Fig. 1) compared to what was observed in mosquitoes fed on noninfected hamsters or hamsters infected with an agametocytic strain of P. berghei (Anka 2.33, obtained from Dr. Robert Sinden of Imperial College, London). Finally, a band electrophoretically indistinguishable from AM2 was detectable in S mosquitoes after either an infective or a noninfective bloodmeal (Fig. 1). There was no evidence of upregulation associated with Plasmodium infection of S mosquitoes. To test the effects of protease inhibitors on the bands, zymography was done as described but with the addition of one of several inhibitors to the washing buffer and the chromagenic substrate. No significant inhibition of the aminopeptidase isozymes was detected after exposure to serine protease inhibitors (antipain–dihydrochloride [74 mM] or aprotinin [0.3 mM], nor to the cystine protease inhibitor E-64 [28 mM]). All bands were inhibited by the Zn21 chelator, 1,10-phenanthroline [10 mM], and by the aminopeptidase inhibitor, bestatin [130 mM]. Thus, all of the isobands met the criteria for being authentic aminopeptidases on the basis of their specificity to aminopeptidase substrates rather than substrates of other proteolytic enzymes (data not shown), their susceptibility to specific inhibitors of aminopeptidase activity and to a Zn21 chelator, and their insensitivity to inhibitors of serine proteases and cysteine proteases. Of particular interest was our observation of a differentially high inhibition of AM2 and the band immediately above it, after exposure to amastatin [10 mM], a specific inhibitor of some aminopeptidases. Our findings with the “immune” aminopeptidase isoband appears to be the first described upregulation of a specific An. gambiae protein in response to infection with a malaria parasite; furthermore, this upregulation was restricted to R mosquitoes. Recent studies have demonstrated upregulation of immune marker RNA’s specifically after a malaria-infected bloodmeal taken by An. gambiae (Dimopoulos et al. 1997; Richman et al. 1997). These studies could not, however, detect any differences between R and S mosquitoes. A potential serine protease candidate for involvement in host defense has been reported from An. gambiae (Han et al. 1997). The gene for this protease did not show increased transcription after a bloodmeal, suggesting its lack of involvement in bloodmeal digestion; moreover, there was greater expression of this gene in R than in S mosquitoes. Aminopeptidases have been shown to have a wide range of functions in both wound healing and host defense against invasive microorganisms. Elevation of aminopeptidase activity has been reported in human burn wound exudates (Prager et al. 1994), in tomato plants infected with Pseudomonas syringae (Pautot et al. 1993), and in strains of An. stephensi genetically selected for resistance to P. falciparum (Feldmann et al. 1990). Aminopeptidase activity is also upregulated in the serum of
Biomphalaria glabrata snails parasitized by the trematode Echinostoma lindoense, and it was suggested that the enzyme may act to uncover cryptic sites on the invader (Cheng et al. 1978). Thus, the striking elevation of AM2 in R mosquitoes 24 h after a P. berghei-infected bloodmeal could conceivably be related to host defense against the malaria parasite. However, the specific role, if any, of this putative “immune” aminopeptidase in a response against the malaria parasite remains to be established. When the aminopeptidase inhibitor bestatin was supplied to tomato plants, Schaller et al. (1995) observed induction of several defenseassociated genes, including the gene for leucine aminopeptidase. They suggested that bestatin may be exerting its effects close to the level of transcriptional control of these defense genes. Using a parallel approach, we fed a related synthetic inhibitor of aminopeptidase (amastatin at 1 mg/ml) within a noninfected hamster bloodmeal presented to R mosquitoes through a parafilm membrane and observed an upregulation of AM2 comparable to that observed following an infected bloodmeal (data not shown), thus suggesting an inhibitor-induced mechanism similar to that described by Schaller et al. (1995). For further studies with AM2, our ability to strikingly inhibit its activity with amastatin presents a powerful tool to investigate the in vivo function of this enzyme and to purify it by affinity chromatography using amastatin attached to agarose beads. (This investigation received financial assistance from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and through N.I.H. National Research Service Award 5T32AI07180-15 0071 from which Ahron Rosenfeld received stipend support as a pre-doctoral fellow.)
REFERENCES
Cheng, T. C., Lie, K. J., Heyneman, D., and Richards C. S. 1978. Elevation of aminopeptidase activity in Biomphalaria glabrata (Mollusca) parasitized by Echinostoma lindoense (Trematoda). Journal of Invertebrate Pathology 31, 57–62. Collins, F. H., Sakai, R. K., Vernick, K. D., Paskewitz, S., Seeley, D. C., Miller, L. H., Collins, W. E., Campbell, C. C., and Gwadz, R. W. 1986. Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 234, 607–610.
FIG. 1. The 8–25% gradient gel Native–PAGE enzymogram of Anopheles gambiae L35 (a) and “wild-type” G3 (b) midgut extract and hemolymph from mosquitoes that had been sugar fed (SF) or received a normal bloodmeal (NBM) or a Plasmodium berghei-infected bloodmeal (IBM) (post-24-h bloodmeal collection). For midgut samples, a total of 0.15 mosquito equivalents (0.4 mg of midgut protein) was loaded per lane. For hemolymph samples, 0.2 mg of hemolymph protein was loaded per lane. Gels were then run for 312 accumulated volt-h at 158C. Substrate used was L-leucine p-nitroanilide for the determination of aminopeptidase-like activity. Arrowhead denotes a band found to be upregulated after an infected bloodmeal in malaria-resistant L3-5 mosquitoes. The midgut aminopeptidase activity detected was determined to be of endogenous origin since controls such as uninfected hamster blood, infected hamster blood, mosquito salivary gland extract, and Malpighian tubule extract failed to yield similar bands when these control materials were applied to the gel in amounts equivalent to the amounts of midgut protein applied per lane. The far-left lane shows molecular mass standards (M): (thyroglobulin [669 kDa], ferritin [440 kDa], catalase [232 kDa], lactate dehydrogenase [140 kDa], and BSA [67 kDa]; Pharmacia). Because migration of nonreduced proteins during Native–PAGE depends upon charge density as well as molecular size of the protein (Rothe and Maurer 1986), the markers cannot be used directly to assess molecular masses of mosquito proteins but rather only as indicators of the relative location of electrophoretically separated proteases.
104 Crampton, J. M. 1994. Approaches to vector control: New and trusted. 3. Prospects for genetic manipulation of insect vectors. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 141–143. Dimopoulos, G., Richman, A., Mu¨ller, H. M., and Kafatos, F. C. 1997. Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites.Proceedings of the National Academy of Science USA 94, 11508–11513. Feldmann, A. M., Billingsley, P. F., and Savelkoul, E. 1990. Bloodmeal digestion by strains of Anopheles stephensi of differing susceptibility to Plasmodium falciparum. Parasitology 101, 193–200. Han, Y. S., Salazar, C. E., Reese-Stardy, S. R., Cornel, A., Gorman, M. J., Collins, F. H., and Paskewitz, S. M. 1997. Cloning and characterization of a serine protease from the human malaria vector, Anopheles gambiae. Insect Molecular Biology 6, 385–395. Ohlsson, B. G., Westro¨m, B. R., and Karlsson, B. W. 1986. Enzymoblotting: A method for localizing proteinases and their zymogens using para-nitroanilide substrates after agarose gel electrophoresis and transfer to nitrocellulose. Analytical Biochemistry 152, 239–244. Pautot, V., Holzer, F. M., Reisch, B., and Walling, L. L. 1993. Leucine aminopeptidase: An inducible component of the defense response in Lycopersicon esculentum (tomato). Proceedings of the National Academy of Science USA 90, 9906–9910. Phastsystem Owners Manual. 1986. Laboratory Separation Division, Pharmacia-LKB, Uppsala, Sweden.
ROSENFELD AND VANDERBERG
Prager, M. D., Sabeh, F., Baxter, C. R., Atiles, L., and Hartline, B. 1994. Dipeptidyl peptidase IV and aminopeptidase in burn wound exudates: implications for wound healing. Journal of Trauma 36, 629–633. Richman A., Dimopoulos, G., Seeley, D., and Kafatos, F. C. 1997. Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes. EMBO Journal 16, 101–106. Rosenfeld A., and Vanderberg J. P. 1998. Identification of electrophoretically-separated proteases from midgut and hemolymph of adult Anopheles stephensi mosquitoes. Journal of Parasitology 84, 361–365. Rothe, G. M., and Maurer, W. D. 1986. One dimensional PAA-gel electrophoretic techniques to separate functional and denatured proteins. In “Gel Electrophoresis of Proteins” (M. J. Dunn, Ed.), pp. 37–140. IOP Publishing Ltd., Bristol, U.K. Schaller, A., Bergey, D. R., and Ryan, C. A. 1995. Induction of wound response gene in tomato leaves by Bestatin, an inhibitor of aminopeptidases. The Plant Cell 7, 1893–1898. Zheng L., Cornel, A. J., Wang, R., Erfle, H., Voss, H., Ansorge, W., Kafatos, F. C., and Collins, F. H. 1997. Quantitative trait loci for refractoriness of Anopheles gambiae to Plasmodium cynomolgi B. Science 276, 425–428. Received 4 November 1998; accepted with revision 9 June 1999