High-frequency burst vagal nerve simulation therapy in a natural primate model of genetic generalized epilepsy.

PURPOSE: Since the approval of Vagal Nerve Stimulation (VNS) Therapy for medically refractory focal epilepsies in 1997, it has been also reported to be effective for a wide range of generalized seizures types and epilepsy syndromes. Instead of conventional VNS Therapy delivered at 20-30Hz signal frequencies, this study evaluates efficacy and tolerability of high-frequency burst VNS in a natural animal model for genetic generalized epilepsy (GGE), the epileptic baboon. METHODS: Two female baboons (B1 P.h. Hamadryas and B2 P.h. Anubis x Cynocephalus) were selected because of frequently witnessed generalized tonic-clonic seizures (GTCS) for VNS implantation. High-frequency burst VNS Therapy was initiated after a 4-5 week baseline; different VNS settings (0.25, 2 or 2.5mA, 300Hz, 4 vs 7 pulses, 0.5-2.5s interburst interval, and intermittent stimulation for 1-2 vs for 24h per day) were tested over the subsequent 19 weeks, which included a 4-6 week wash-out period. GTCS frequencies were quantified for each setting, while seizure duration and postictal recovery times were compared to baseline. Scalp EEG studies were performed at almost every setting, including intermittent light stimulation (ILS) to evaluate photosensitivity. Pre-ILS ictal and interictal discharge rates, as well as ILS responses were compared between trials. The Novel Object test was used to assess potential treatment effects on behavior. RESULTS: High-frequency burst VNS Therapy reduced GTCS frequencies at all treatment settings in both baboons, except when output currents were reduced (0.25mA) or intermittent stimulation was restricted (to 1-2h/day). Seizure duration and postictal recovery times were unchanged. Scalp EEG studies did not demonstrate treatment-related decrease of ictal or interictal epileptic discharges or photosensitivity, but continuous treatment for 120-180s during ILS appeared to reduce photoparoxysmal responses. High-frequency burst VNS Therapy was well-tolerated by both baboons, without cardiac or behavioral changes. Repetitive muscle contractions involving the neck and left shoulder girdle were observed intermittently, most commonly at 0.5 interburst intervals, but these were transient, resolving with a few cycles of stimulation and not noted in wakefulness. CONCLUSIONS: This preclinical pilot study demonstrates efficacy and tolerability of high-frequency burst VNS Therapy in the baboon model of GGE. The muscle contractions may be due to aberrant propagation of the stimulus along the vagal nerve or to the ansa cervicalis, but can be reduced by minimal adjustment of current output or stimulus duration.

Effect of aging on regional cerebral blood flow responses associated with osmotic thirst and its satiation by water drinking: a PET study.

Levels of thirst and ad libitum drinking decrease with advancing age, making older people vulnerable to dehydration. This study investigated age-related changes in brain responses to thirst and drinking in healthy men. Thirst was induced with hypertonic infusions (3.1 ml/kg 0.51M NaCl) in young (Y) and older (O) subjects. Regional cerebral blood flow (rCBF) was measured with positron emission tomography (PET). Thirst activations were identified by correlating rCBF with thirst ratings. Average rCBF was measured from regions of interest (ROI) corresponding to activation clusters in each group. The effects of drinking were examined by correlating volume of water drunk with changes in ROI rCBF from maximum thirst to postdrinking. There were increases in blood osmolality (Y, 2.8 +/- 1.8%; O, 2.2 +/- 1.4%) and thirst ratings (Y, 3.1 +/- 2.1; O, 3.7 +/- 2.8) from baseline to the end of the hypertonic infusion. Older subjects drank less water (1.9 +/- 1.6 ml/kg) than younger subjects (3.9 +/- 1.9 ml/kg). Thirst-related activation was evident in S1/M1, prefrontal cortex, anterior midcingulate cortex (aMCC), premotor cortex, and superior temporal gyrus in both groups. Postdrinking changes of rCBF in the aMCC correlated with drinking volumes in both groups. There was a greater reduction in aMCC rCBF relative to water drunk in the older group. Aging is associated with changes in satiation that militate against adequate hydration in response to hyperosmolarity, although it is unclear whether these alterations are due to changes in primary afferent inflow or higher cortical functioning.

Effect of 30 per cent maternal nutrient restriction from 0.16 to 0.5 gestation on fetal baboon kidney gene expression.

Previous studies in rodents and sheep show that maternal nutrient restriction during pregnancy alters fetal renal development. To date, no studies using fetal baboon RNA with human Affymetrix gene chips have been published. In the present study we have (1) evaluated the specificity of the Affymetrix human gene array 'Laboratory on a Chip' system for use with fetal baboon mRNA and (2) investigated the effects of moderate maternal global nutrient restriction (NR; 70% of ad libitum animals) from early (30 days gestation (dG)) to mid-gestation (90 dG; term = 184 dG) on the fetal baboon kidney. Morphometric and blood measurements were made on 12 non-pregnant baboons before they were bred. All baboons were fed ad libitum until 30 days pregnant, at which time six control baboons continued to feed ad libitum (control - C) while six received 70% of the C diet on a weight adjusted basis. Fetal kidneys were collected following caesarean section at 90 dG, with samples flash frozen and fixed for histological assessment. Fetal hip circumference was decreased in the NR group (68 +/- 2 versus 75 +/- 2 mm), while fetal body weight and all other measurements of fetal size were not different between C and NR at 90 dG. Maternal body weight was decreased in the NR group (12.16 +/- 0.34 versus 13.73 +/- 0.55 kg). Having established the specificity of the Affymetrix system for fetal baboon mRNA, gene expression profiling of fetal kidneys in the context of our maternal nutrient restriction protocol shows that NR resulted in a down-regulation of genes in pathways related to RNA, DNA and protein biosynthesis, metabolism and catabolism. In contrast, genes in cell signal transduction, communication and transport pathways were up-regulated in the NR group. These changes indicate that even a moderate level of maternal global NR impacts fetal renal gene pathways. Our histological assessment of renal structure indicates decreased tubule density within the cortex of NR kidneys compared with controls. The number of glomerular cross-sections per unit area were unaffected by NR, suggesting that tubule tortuosity and/or tubule length was decreased in the NR kidney. Taken together the changes indicate that NR results in accelerated fetal renal differentiation. The negative impact of poor maternal nutrition on the fetal kidney may therefore be in part due to shortening of critical phases of renal growth resulting in decreased functional capacity in later life. These findings may have important implications for postnatal renal function, thereby contributing to the observed increased predisposition to hypertension and renal disease in the offspring of nutrient restricted mothers.

Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests.

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor.

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371-379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN-free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN-adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN-induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L-like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN-degrading protease activity.

Synergy between angiotensin and aldosterone in evoking sodium appetite in baboons.

The synergy between ANG II and aldosterone (Aldo) in the induction of salt appetite, extensively studied in rats, has been tested in baboons. ANG II was infused intracerebroventricularly at 0.5 or 1.0 microg/h; Aldo was infused subcutaneously at 20 microg/h. Separate infusions over 7 days had no significant effect on the daily intake of 300 mM NaCl. Concurrent infusions, however, increased daily NaCl intake approximately 10-fold and daily water intake approximately 2.5-fold. In addition, the combined infusions caused 1) a reduction in daily food intake, 2) changes in blood composition indicative of increased vasopressin release, and 3) changes of urinary excretion rates of cortisol and Aldo indicative of increased ACTH release. Arterial blood pressure, measured in two baboons, rose during concurrent ANG II and Aldo treatment. These results indicate a potent synergy between central ANG II and peripheral Aldo in stimulating salt appetite in baboons. At the same time, other ANG II-specific brain mechanisms concerned with water intake, food intake, vasopressin release, ACTH release, and blood pressure regulation appear to have been activated by the same type of synergy. These central enhancement processes have never been previously demonstrated in primates.

Ingestive responses to administration of stress hormones in baboons.

Experimental stress and the administration of the stress hormone ACTH have been reported to stimulate sodium appetite in many nonprimate species. Experiments were conducted to determine whether prolonged intracerebroventricular infusions of the neuropeptides corticotropin-releasing factor (CRF) and urocortin (Ucn), or systemic administration of ACTH, affected ingestive behaviors in a nonhuman primate, the baboon. Intracerebroventricular infusions of CRF or Ucn significantly decreased daily food intake. The decrease with Ucn continued into the postinfusion period. These infusions did not alter daily water intake. Daily voluntary intake of 300 mM NaCl solution was not increased, and there was evidence of reductions on days 2-4 of the infusions. Intramuscular injections of porcine ACTH or synthetic ACTH (Synacthen) for 5 days did not affect daily NaCl intake, although the doses were sufficient to increase cortisol secretion and arterial blood pressure. Sodium depletion by 3 days of furosemide injections did induce a characteristic sodium appetite in the same baboons. These results demonstrate the anorexigenic action of CRF and Ucn in this primate. Also, CRF, Ucn, and ACTH did not stimulate sodium appetite at the doses used.

Possible contribution of brain angiotensin III to ingestive behaviors in baboons.

Recent experiments with specific aminopeptidase inhibitors in rats have strengthened earlier proposals that ANG III may be an important regulatory peptide in the brain. Central mechanisms regulating blood pressure, ingestive behaviors, and vasopressin release could be involved. Arguments in favor of a role for ANG III depend, in part, on the efficacy of ANG III as an agonist. These first studies in primates tested whether ANG III stimulates ingestive behaviors in baboons. Intracerebroventricular (ICV) infusions of ANG III were as potent as ANG II in stimulating water drinking and intake of NaCl solution. On the basis of this criterion and consistent with findings in rats, ANG III could be a main effector peptide in the regulation of ingestive behaviors in a primate.

Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase.

Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010-1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 x 107 M-1sec-1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

Sodium-lithium countertransport activity is linked to chromosome 5 in baboons.

The genes involved in the regulation of cellular sodium transport characteristics, which are correlated with some forms of essential hypertension, have not yet been identified. We are studying the genes and environmental factors that affect red blood cell sodium-lithium countertransport (SLC) activity and intracellular sodium (ICNa) concentration in 634 baboons that comprise 11 pedigrees of 2 and 3 generations each. To detect and locate possible quantitative trait loci (QTLs) that affect SLC activity and ICNa concentration, we performed a genome screen by using a maximum likelihood-based variance-components linkage analysis program (SOLAR). SLC and ICNa phenotypes as well as genotypes on 281 microsatellite loci were available for all pedigreed animals. Both SLC and ICNa traits were highly heritable (residual heritability 0.593+/-0.083 [P<0.0001] and 0.739+/-0.082 [P<0.0001], respectively). We obtained evidence that a possible QTL for SLC activity is located on the baboon homologue of human chromosome 4 between D4S2456 and D4S2365 with a maximum multipoint lod score of 9.3 (P<10(-)(10)) near D4S1645. This QTL accounts for approximately two thirds of the total additive genetic variation in SLC activity in baboons. Although ICNa concentration was highly heritable, we found no evidence for linkage to a QTL with use of this methodology. Thus, we have evidence that a gene located on the baboon homologue of human chromosome 4 (baboon chromosome 5) affects cell sodium transport in baboons.

Neuroimaging of cerebral activations and deactivations associated with hypercapnia and hunger for air.

There are defined medullary, mesencephalic, hypothalamic, and thalamic functions in regulation of respiration, but knowledge of cortical control and the elements subserving the consciousness of breathlessness and air hunger is limited. In nine young adults, air hunger was produced acutely by CO(2) inhalation. Comparisons were made with inhalation of a N(2)/O(2) gas mixture with the same apparatus, and also with paced breathing, and with eyes closed rest. A network of activations in pons, midbrain (mesencephalic tegmentum, parabrachial nucleus, and periaqueductal gray), hypothalamus, limbic and paralimbic areas (amygdala and periamygdalar region) cingulate, parahippocampal and fusiform gyrus, and anterior insula were seen along with caudate nuclei and pulvinar activations. Strong deactivations were seen in dorsal cingulate, posterior cingulate, and prefrontal cortex. The striking response of limbic and paralimbic regions points to these structures having a singular role in the affective sequelae entrained by disturbance of basic respiratory control whereby a process of which we are normally unaware becomes a salient element of consciousness. These activations and deactivations include phylogenetically ancient areas of allocortex and transitional cortex that together with the amygdalar/periamygdalar region may subserve functions of emotional representation and regulation of breathing.

Brain responses associated with consciousness of breathlessness (air hunger).

Little is known about the physiological mechanisms subserving the experience of air hunger and the affective control of breathing in humans. Acute hunger for air after inhalation of CO(2) was studied in nine healthy volunteers with positron emission tomography. Subjective breathlessness was manipulated while end-tidal CO(2-) was held constant. Subjects experienced a significantly greater sense of air hunger breathing through a face mask than through a mouthpiece. The statistical contrast between the two conditions delineated a distributed network of primarily limbic/paralimbic brain regions, including multiple foci in dorsal anterior and middle cingulate gyrus, insula/claustrum, amygdala/periamygdala, lingual and middle temporal gyrus, hypothalamus, pulvinar, and midbrain. This pattern of activations was confirmed by a correlational analysis with breathlessness ratings. The commonality of regions of mesencephalon, diencephalon and limbic/paralimbic areas involved in primal emotions engendered by the basic vegetative systems including hunger for air, thirst, hunger, pain, micturition, and sleep, is discussed with particular reference to the cingulate gyrus. A theory that the phylogenetic origin of consciousness came from primal emotions engendered by immediate threat to the existence of the organism is discussed along with an alternative hypothesis by Edelman that primary awareness emerged with processes of ongoing perceptual categorization giving rise to a scene [Edelman, G. M. (1992) Bright Air, Brilliant Fire (Penguin, London)].

Neuroimaging evidence implicating cerebellum in the experience of hypercapnia and hunger for air.

Recent neuroimaging and neurological data implicate cerebellum in nonmotor sensory, cognitive, vegetative, and affective functions. The present study assessed cerebellar responses when the urge to breathe is stimulated by inhaled CO(2). Ventilation changes follow arterial blood partial pressure CO(2) changes sensed by the medullary ventral respiratory group (VRG) and hypothalamus, entraining changes in midbrain, pons, thalamus, limbic, paralimbic, and insular regions. Nearly all these areas are known to connect anatomically with the cerebellum. Using positron emission tomography, we measured regional brain blood flow during acute CO(2)-induced breathlessness in humans. Separable physiological and subjective effects (air hunger) were assessed by comparisons with various respiratory control conditions. The conjoint physiological effects of hypercapnia and the consequent air hunger produced strong bilateral, near-midline activations of the cerebellum in anterior quadrangular, central, and lingula lobules, and in many areas of posterior quadrangular, tonsil, biventer, declive, and inferior semilunar lobules. The primal emotion of air hunger, dissociated from hypercapnia, activated midline regions of the central lobule. The distributed activity across the cerebellum is similar to that for thirst, hunger, and their satiation. Four possible interpretations of cerebellar function(s) here are that: it subserves implicit intentions to access air; it provides predictive internal models about the consequences of CO(2) inhalation; it modulates emotional responses; and that while some cerebellar regions monitor sensory acquisition in the VRG (CO(2) concentration), others influence VRG to adjust respiratory rate to optimize partial pressure CO(2), and others still monitor and optimize the acquisition of other sensory data in service of air hunger aroused vigilance.

A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera).

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.

Neuroimaging evidence implicating cerebellum in support of sensory/cognitive processes associated with thirst.

Recent studies implicate the cerebellum, long considered strictly a motor control structure, in cognitive, sensory, and affective phenomenon. The cerebellum, a phylogenetically ancient structure, has reciprocal ancient connections to the hypothalamus, a structure important in vegetative functions. The present study investigated whether the cerebellum was involved in vegetative functions and the primal emotions engendered by them. Using positron emission tomography, we examined the effects on the cerebellum of the rise of plasma sodium concentration and the emergence of thirst in 10 healthy adults. The correlation of regional cerebral blood flow with subjects' ratings of thirst showed major activation in the vermal central lobule. During the development of thirst, the anterior and posterior quadrangular lobule, lingula, and the vermis were activated. At maximum thirst and then during irrigation of the mouth with water to alleviate dryness, the cerebellum was less activated. However, 3 min after drinking to satiation, the anterior quadrangular lobule and posterior cerebellum were highly activated. The increased cerebellar activity was not related to motor behavior as this did not occur. Instead, responses in ancient cerebellar regions (vermis, fastigal nucleus, archicerebellum) may be more directly related to vegetative and affective aspects of thirst experiences, whereas activity in neocerebellar (posterior) regions may be related to sensory and cognitive aspects. Moreover, the cerebellum is apparently not involved in the computation of thirst per se but rather is activated during changes in thirst/satiation state when the brain is "vigilant" and is monitoring its sensory systems. Some neocerebellar activity may also reflect an intentionality for gratification by drinking inherent in the consciousness of thirst.

Neuroimaging of genesis and satiation of thirst and an interoceptor-driven theory of origins of primary consciousness.

There are defined hypothalamic functions in the genesis of thirst, but little is known of the cortical processes subserving consciousness of thirst notwithstanding the medical disorders that occur in psychiatric illness, addiction, and the attested decline of thirst with aging. In 10 adult males, positron emission tomography scans were made (i) during genesis of moderate thirst by infusion of i.v. hypertonic saline 0.51 M, (ii) after irrigation of the mouth with water to remove the sensation of dryness, and (iii) 3, 14, 45, and 60 minutes after drinking water to fully satiate thirst. The correlation of regional cerebral blood flow with thirst score showed the major activation to be in the posterior cingulate. Maximum thirst sensation evoked 13 highly significant activations and 9 deactivations in cingulate and parahippocampal gyri, insula, thalamus, amygdala, and mesencephalon. It is possible that cingulate sites (Brodmann's areas 32, 24, and 31) that persisted with wet mouth but disappeared immediately after drinking to satiation may have an important role in the consciousness of thirst. Consciousness of thirst, a primal vegetative emotion, and satiation of thirst appear to be subserved by phylogenetically ancient brain regions. This is salient to current discussion on evolutionary emergence of primary consciousness.

Correlation of regional cerebral blood flow and change of plasma sodium concentration during genesis and satiation of thirst.

Positron emission tomography studies were conducted during genesis of moderate thirst by rapid i.v. infusion of hypertonic saline (0.51 M) and after satiation of thirst by drinking water. The correlation of regional cerebral blood flow with the change in the plasma Na concentration showed a significant group of cerebral activations in the anterior cingulate region and also a site in the middle temporal gyrus and in the periaqueductal gray. Strongest deactivations occurred in the parahippocampal and frontal gyri. The data are consistent with an important role of the anterior cingulate in the genesis of thirst.

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II.

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.