Molecular evolution of Bowman-Birk type proteinase inhibitors in flowering plants

The Bowman-Birk family (BBI) of proteinase inhibitors is probably the most studied family of plant inhibitors. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the GenBank database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events.     

Adult Colorado potato beetles, Leptinotarsa decemlineata compensate for nutritional stress on oryzacystatin I-transgenic potato plants by hypertrophic behavior and over-production of insensitive proteases

Protease inhibitors have been proposed as potential control molecules that could be engineered into potato plants for developing crops resistant to the Colorado potato beetle, Leptinotarsa decemlineata, a major pest of potato and other Solanaceae. In this study, we examined the effects of feeding young female beetles with foliage from a cultivar of the "Kennebec" potato line (K52) transformed with a gene encoding oryzacystatin I (OCI), a specific cysteine proteinase inhibitor with proven activity against cathepsin H-like enzymes of larvae and adults of the potato beetle. To evaluate the insect's performance, we collected data over a 16-d postemergence period on survival, diapause incidence, foliage consumption, weight gain, and oviposition of females. Tested individuals were fed untransformed (control) and OCI-transformed foliage at two stages of potato leaf differentiation, corresponding to "low" and "high" levels of OCI expression in leaves of K52. The OCI-expressing foliage did not affect female survival (close to 100%), incidence of diapause (15-30%), relative growth rate (RGR) during postemergence growth (5-9% d(-1)) or maximum weight reached (140-160 mg). Neither did it affect female reproductive fitness as measured by preoviposition time (8-9 d), 16-d fecundity (220-290 eggs), or egg eclosion incidence (86-91%). However, nutritional stress to females feeding on OCI foliage was evident, as reflected in their lower efficiency of conversion of ingested foliage (ECI) during postemergence growth, increased foliage consumed per egg laid (up to 119% more), and adaptation of their digestive proteolytic system to the inhibitory effect of OCI. Interestingly, beetles fed foliage expressing the highest level of OCI reacted rapidly to the presence of OCI by producing OCI-insensitive proteases, and exhibiting strong hypertrophic behavior by ingestion of 2.4-2.5 times more OCI rich foliage apparently as a compensatory response for nutritional stress due to the protease inhibitor in their diet.     

Identification of a soluble enzyme from C3H/10T1/2 cells which is inhibited by the Bowman-Birk proteinase inhibitor

The anticarcinogenic Bowman-Birk proteinase inhibitor (BBI) inhibits a 70-kDa serine proteinase in C3H/10T1/2 transformed fibroblasts. Two serine proteinases, the proline endopeptidase and a novel neutral proteolytic activity, both having a mass of approximately 70-kDa, were isolated from the cytoplasm of C3H/10T1/2 cells. BBI did not inhibit diisopropylfluorophosphate binding to the proline endopeptidase or its ability to hydrolyze peptides. However, BBI blocked the binding of diisopropylfluorophosphate and inhibited the cleavage of peptides by the novel cytoplasmic enzyme. Thus BBI does not inhibit the proline endopeptidase but another soluble 70-kDa serine proteinase from C3H/10T1/2 cells.     

Long-term effects of soybean protease inhibitors on digestive enzymes, survival and learning abilities of honeybees

We have investigated the effects of long-term ingestion of two serine proteinase inhibitors (PIs), the Kunitz Soybean trypsin inhibitor (SBTI) and the Bowman-Birk inhibitor (BBI) on survival, learning abilities involved in the foraging behaviour, and digestive physiology of the honeybee (Apis mellifera L., Hymenoptera). A threshold-dose was established, above which adverse effects of long-term ingestion of the PIs tested are to be expected. The experiments reported herein could be extended to other PIs or gene products used to confer insect resistance, and be part of a general procedure used to assess the innocuousness of transgenic melliferous plants to honeybees.     

Synthesis of active oryzacystatin I in transgenic potato plants

Summary Transformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.Abbreviations CPB Colorado potato beetle - E-64 trans-epoxy-succinyl-L-leucylamido (4-guanidino) butane - OCI oryzacystatin I - PI proteinase inhibitor - PMSF phenylmethylsulfonyl fluoride     

Toxicity toChrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor

The aim of this study was to test the potential of proteinase inhibitors to controlChrysomela tremulae, a beetle that causes severe damage in young plantations and in short-rotation intensive culture (SRIC) of poplar. As a first step, cysteine proteinases were determined to be the major digestive proteinases ofC. tremulae and oryzacystatin OCI, a cysteine proteinase inhibitor, was shown to inhibit this activityin vitro. The gene encoding OCI was introduced into poplar (Populus tremula ×P. tremuloides) and transgenic plants expressing OCI at a high level were selected. Feeding tests on these transgenic plants demonstrate the toxicity of OCI-producing poplar leaves againstC. tremulae larvae.J.C. Leplé and M. Bonadé-Bottino contributed equally to the research presented in this paper.     

Structure-function relationship of bromelain isoinhibitors from pineapple stem

Bromelain isoinhibitors from pineapple stem (BIs) are unique double-chain inhibitors and inhibit the cysteine proteinase bromelain competitively. The three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded anti-parallel beta-sheet. Unexpectedly, BIs were found to share similar folding and disulfide-bond connectivities not with the cystatin superfamily, but with Bowman-Birk trypsin/chymotrypsin inhibitor (BBI). The structural similarity between them suggests that BIs and BBI have evolved from a common ancestor and differentiated in function during the course of molecular evolution.     

Changes in midgut endopeptidase activity of Spodoptera frugiperda (Lepidoptera: Noctuidae) are responsible for adaptation to soybean proteinase inhibitors

The development of transgenic maize plants expressing soybean proteinase inhibitors could reduce the economic damage of one of the major maize pests in Brazil, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797). We examined the influence of soybean proteinase inhibitors on digestive enzyme properties and development of S. frugiperda larvae. The inhibition of trypsin and chymotrypsin activities in vitro by soybean proteinase inhibitors suggested that either Kunitz (SBTI) or Bowman-Birk (SBBI) would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of semipurified soybean inhibitors did not result in a significant reduction of growth and development of fall armyworm. Therefore, digestive serine proteinase activities (trypsin and chymotrypsin) of fall armyworm larvae were characterized. The results suggest that S. frugiperda was able to physiologically adapt to dietary proteinase inhibitors by altering the complement of proteolytic enzymes in the insect midguts.     

Growth stimulation of beetle larvae reared on a transgenic oilseed rape expressing a cysteine proteinase inhibitor

The resistance of a transgenic line of oilseed rape expressing constitutively the cysteine proteinase inhibitor oryzacystatin I (OCI) was assessed against Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). The levels of OCI expression in the transformed line averaged 0.2% and 0.05% of total soluble protein in leaves and petioles respectively. In vitro analyses showed that P. chrysocephala larvae use both cysteine and serine proteinases for protein digestion, and that all the cysteine proteolytic activity is OCI-sensitive. However, bioassays showed that adults fed identically on leaf discs from control or transformed plants. When larvae were reared on transgenic plants expressing OCI, they showed an increase in weight gain compared to those reared on control plants. Furthermore, those larvae from transgenic plants exhibited a 2-fold increase in both cysteine and serine proteolytic activity as a reponse to the presence of OCI. The plasticity of insect digestive physiology and feeding behaviour are discussed, as well as the relevance of engineering a genotype expressing both types of proteinase inhibitors.     

Potential effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid parasitoid Aphidius ervi Haliday (Hymenoptera, Braconidae)

Protease inhibitors (PIs) have been shown to cause lethal and sublethal effects on aphids depending on the kind of PI and aphid species. Therefore, these proteins might affect aphid parasitoids directly by inhibiting their digestive proteolysis or indirectly via their development in a less suitable host. In our study, the risk of exposure and the potential effects of soybean Bowman-Birk inhibitor (SbBBI) and oryzacystatin I (OCI) on the aphid endoparasitoid Aphidius ervi were investigated using artificial diet to deliver PIs. Immunoassays showed that both SbBBI and OCI were detected in the honeydew of aphids reared on artificial diet containing these recombinant proteins at 100 microg/mL. However, only SbBBI was detected in parasitoid larvae, while this PI could not be detected in adult parasitoids emerged from PI-intoxicated aphids. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of A. ervi predominantly relies on serine proteases and especially on chymotrypsin-like activity. Bioassays using SbBBI and OCI on artificial diet were performed. A. ervi that developed on intoxicated aphids had impaired fitness. Thus development and parasitism success of parasitoids exposed to OCI were severely affected. On the contrary, SbBBI only altered significantly female size and sex ratio. Direct exposure to PIs through adult food intake did not affect female's longevity, while SbBBI and OCI (100 microg/mL) induced 69% and 30% inhibition of digestive protease activity, respectively. These studies made it possible to estimate the risk of exposure to plant PIs and the sensitivity of the aphid parasitoid A. ervi to these entomotoxins, by combining immunological, biochemical and biological approaches. First it pointed out that only immature stages are affected by PIs. Secondly, it documented two different modes of effect, according to the nature of the PIs and both host and parasitoid susceptibility. OCI prevented the development of A. ervi mainly due to the host susceptibility, whereas SbBBI only induced sublethal effects on the parasitoid, possibly due to both direct action on the parasitoid susceptible proteases, and host-mediated action through size reduction.     

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Draga?evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.     

Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressing oryzacystatin I

The aim of this study was to assess the potential effect of a transgenic line of oilseed rape expressing oryzacystatin I (OCI) on two strains of cabbage seed weevil. The level of OCI expression in seeds was approximately 0.05% of total soluble proteins. The insects were field-collected in two different locations, and their progeny was analyzed after a 3 week-development in pods. Both strains showed a similar pattern of proteolytic activity, and similar levels of OCI-sensitive proteinase activity in vitro. However, the larvae showed differential susceptibility to the transgenic plants. Despite inhibition of digestive proteinases in vitro by OCI in both strains, one strain showed an increased growth rate when fed the transgenic seeds, while the other strain remained unaffected. While suggesting the importance of studying individuals from different populations when assessing the effect of proteinase inhibitor-expressing plants on insect growth, our results also point out the necessity of studying the biochemical interactions taking place in vivo between the recombinant inhibitors and their target proteinases.     

Liposomal formulations of protein proteinase inhibitors: Preparation and specific activity

Possibility of encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) was investigated. The influence of the PC/PE ratio (w/w) on efficiency of incorporation of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and also between bilayers. The study of biological (antitrypsin and antichymotrypsin) activity demonstrated partial spatial shielding of active sites of proteins entrapped in liposomes. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI is consistent with this hypothesis and suggests that this shielding is reversible. Stability of liposomes was examined using three various media modeling gastrointestinal fluids (gastric and intestinal juices and fluids). Data obtained indicate that the prepared liposomes seem to be promising formulations for BBI and BPTI delivery.     

Binding of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine alpha-chymotrypsin and bovine beta-trypsin. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C(p), F-T(p) and F-T(t), respectively) to bovine alpha-chymotrypsin (alpha-chymotrypsin) and bovine beta-trypsin (beta-trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: alpha-chymotrypsin: BBI approximately beta-trypsin:BBI approximately beta-trypsin:F-T(t) approximately beta-trypsin:F-T(p) much greater than alpha-chymotrypsin:F-C(p). F-C(p), F-T(p) and F-T(t) do not inhibit beta-trypsin and alpha-chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F-C(p), F-T(p) and/or F-T(t) binding to alpha-chymotrypsin and beta-trypsin decrease, thus reflecting the acid-pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F-C(p), F-T(p) and F-T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.     

Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice

Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage in fact being inferior to free water for short-term survival of nonfeeding first-instar larvae. These results support the view that OCI, an effective inhibitor of a substantial fraction of digestive enzymatic potential in P. bioculatus, should not interfere with its predation potential when expressed in potato plants fed to its prey at a maximum level of approximately 0.8% of total soluble proteins in mature foliage.     

Does host range influence susceptibility of herbivorous insects to non-host plant proteinase inhibitors?

We examined the influence of proteinase inhibitors on digestive enzymes and development of oriental beetle,Exomala orientalis Waterhouse, European chafer,Rhizotrogus majalis (Razoumowsky),Phyllophaga white grub,Phyllophaga anxia (LeConte), cranberry root grub,Lichnanthe vulpina (Hentz), Japanese beetle,Popillia japonica Newman, Asiatic garden beetle, Maladera castanea (Arrow) (Coleoptera: Scarabaeidae), and the black cutworm,Agrotis ipsilon (Rottemburg) (Lepidoptera: Noctuidae). We demonstrated that all species within our test group had alkaline midguts that contained proteinase activity that could be inhibited,in vitro with serine proteinase inhibitors. Our data suggests that host range may influence the susceptibility to non-host inhibitors. Chronic ingestion of the serine proteinase inhibitor, Kunitz-soybean trypsin inhibitor (STI), significantly reduced proteolytic activityin vivo in those species with relatively specialized feeding habits (i.e., cranberry root grub, Japanese beetle, Asiatic garden beetle, and black cutworm). Chronic ingestion of STI also resulted in reduced larval growth and delayed pupation for black cutworm, and elevated larval mortality for Japanese beetle. However, chronic ingestion of STI did not influence larval survival for those species with relatively generalized feeding habits (i.e., oriental beetle, European chafer). Based on these results, we propose mechanistically-based criteria for selecting proteinase inhibitors for phytochemical defense against herbivorous insects.     

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.     

Coupling diet quality and Bowman-Birk and Kunitz-type soybean proteinase inhibitor effectiveness to Diatraea saccharalis development and mortality

We used bioassays to investigate the effect of Bowman‐Birk and Kunitz‐type soybean proteinase inhibitors on two artificial diets (diets 1 and 2) which are commonly used to feed laboratory colonies of larvae of the moth Diatraea saccharalis, monitoring food intake and utilization, and larval development and mortality. Diet 1 was less nutritious, with a low protein content and reduced mineral and essential amino acid (e.g., cysteine, lysine, and methionine) content, while diet 2 was richer and more complete. When proteinase inhibitors were incorporated into the artificial diets, the effects on larval development were significantly greater for those larvae fed diet 1, with the chronic ingestion of proteinase inhibitors reducing the level of trypsin‐like activity in the midgut of larvae fed this diet. Larvae fed diet 2 also showed a reduced level of tryptic activity in the midgut, but this was less marked than for diet 1. These results indicate that despite their inhibitory effect on midgut enzymes, the effectiveness of proteinase inhibitors is directly dependent on the quality of the diet. The different effects seen on insect biology when proteinase inhibitors are added to rich or poor diets suggests that the role of anti‐nutritional proteins in the control of insects might not be adequately addressed by bioassays based on the incorporation of inhibitors into artificial diets.     

Fitness and feeding are affected in the two-spotted stinkbug,Perillus bioculatus,by the cysteine proteinase inhibitor,oryzacystatin I

Plant resistance to insect pests based on recombinant proteinase inhibitors (Pis) could interfere with natural enemies of target pests, as their own proteolytic systems may also be sensitive to large spectrum PIs. Oryzacystatin I (OCI) is a potential insect pest resistance factor currently engineered into a variety of crop plants, including potato Solanum tuberosum. Potential for OCI interfering with female reproduction in Perillus bioculatus, a stinkbug predator of Colorado potato beetle, Leptinotarsa decemlineata, was studied by chronic feeding for 18 days on prey loaded with 1–16 μg OCI/day. Mortality of treated females was negligible, but fertility was reduced by up to 50%. Additional dose-dependent effects in reproducing females included delayed oviposition, reduced fecundity, lower egg mass size, and reduced egg eclosion incidence. Females fed for 18 days on OCI at ≤4 μg/day returned to normal oviposition when switched to prey without OCI after 18 days of treatment, but negative effects persisted for at least 10 days at higher doses. Affected reproduction in P. bioculatus is consistent with the use of OCI-sensitive digestive proteinases by this stinkbug. However, azocaseinase activity in whole body extracts of OCI-fed females increased about twofold indicating compensation, and OCI-sensitive proteinases were still present in extracts. When timed for delay to trigger attack on Colorado potato beetle larvae under controlled conditions, stinkbugs feeding on OCI appeared consistently hungrier than controls fed at similar rate, suggesting that predation by stinkbugs exposed to OCI-recombinant foliage would be higher than normal. Arch. Insect Biochem. Physiol. 38:74–83, 1998. © 1998 Wiley-Liss, Inc.