N-beta-Alanyldopamine: Major Role in Insect Cuticle Tanning.

N-beta-Alanyldopamine is the major tyrosine metabolite in the hemolymph and cuticle during pupal tanning in the tobacco hornworm, Manduca sexta L. Its concentration in hemolymph increases over 800-fold above larval levels by the start of tanning and decreases as the pupal cuticle darkens and hardens. It is a major catechol in species representing several insect orders and is the preferred substrate for pupal cuticular o-diphenol oxidase. In insects, N-beta-alanyldopamine appears to be the main precursor for tanning chemicals at certain developmental stages.

Transgenic avidin maize is resistant to storage insect pests.

Avidin is a glycoprotein found in chicken egg white, that sequesters the vitamin biotin. Here we show that when present in maize at levels of > or =100 p.p.m., avidin is toxic to and prevents development of insects that damage grains during storage. Insect toxicity is caused by a biotin deficiency, as shown by prevention of toxicity with biotin supplementation. The avidin maize is not, however, toxic to mice when administered as the sole component of their diet for 21 days. These dates suggest that avidin expression in food or feed grain crops can be used as a biopesticide against a spectrum of stored-produce insect pests.

Oxidative decarboxylation of 3,4-dihydroxymandelic acid to 3,4-dihydroxybenzaldehyde: electrochemical and HPLC analysis of the reaction mechanism.

Cyclic voltammetric and chronoamperometric data are consistent with a process in which 3,4-dihydroxymandelic acid (DOMA) is oxidized initially in a two-electron step to its corresponding o-benzoquinone. This species is unstable and undergoes the rate-determining loss of CO2 (k = 1.6 s-1 at pH 6 and 25 degrees C) to give an unobserved p-benzoquinone methide intermediate that rapidly isomerizes to 3,4-dihydroxybenzaldehyde (DOBAL), DOBAL is also electroactive at the applied potential and is oxidized in a two-electron step to 4-formyl-1,2-benzoquinone. Subsequent reactions of 4-formyl-1,2-benzoquinone include the oxidation of unreacted DOMA and the hydration of its aldehyde functional group. Oxidation of DOMA directly to its p-benzoquinone methide apparently does not occur. Derivatives of mandelic acid (e.g., 4-hydroxymandelic acid) that are expected to give only their corresponding p-benzoquinone methides upon oxidation afford redox behavior that differs distinctly from that for DOMA.

The mandibular repositioning device: role in the treatment of obstructive sleep apnea.

The role of oral appliances in the routine treatment of obstructive sleep apnea (OSA) is not well defined. This prospective study attempts to clarify the clinical role of a specific oral appliance, the mandibular repositioning device (MRD). This study evaluated the demographic, polysomnographic, and cephalometric radiographic findings predictive of treatment success or failure with the MRD. Twenty-nine patients were diagnosed with mild to severe OSA by nocturnal polysomnography. The majority of these patients were intolerant to nasal continuous positive airway pressure (CPAP) and all were fitted with a MRD. Twenty-three of these patients were compliant initially with MRD use and received post-treatment nocturnal polysomnogrpahy at a mean of 104 days after receiving the device. The respiratory disturbance index (RDI) decreased with MRD use (37 +/- 23 versus 18 +/- 20 events/hour, p < 0.001), and 16 of the 23 patients (69%) were considered responders (decrease in RDI > or = 50% and posttreatment RDI < or = 20). Measurements of subjective and objective daytime sleepiness, nocturnal oxygen desaturation, and snoring were all improved with MRD use. A pre-treatment RDI > 40 was present in four of the seven (67%) non-responders. Age, body mass index, and cephalometric radiographic measurements were not predictive of treatment outcome. Sixteen of 23 patients (70%) continue to use the MRD after 3.4 +/- 0.7 years. This study suggests that the MRD is useful in the long-term treatment of patients with OSA of mild to moderate severity.

Characterization of two high molecular weight catechol-containing glycoproteins from pharate pupal cuticle of the tobacco hornworm, Manduca sexta.

Two high molecular weight cuticular proteins (MSCP120 and MSCP246) were extracted in acidic guanidine hydrochloride solution from tanning abdominal cuticle of Manduca sexta pharate pupae and purified by size exclusion high performance liquid chromatography. The apparent molecular weights were ca. 120 and 246 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both proteins contained high levels of glutamate/glutamine, glycine, serine, alanine and aspartate/asparagine. MSCP120 was enriched in histidine relative to MSCP246, but the reverse was true for valine and proline. Small quantities of beta-alanine and 3,4-dihydroxyphenylalanine (DOPA), as well as other catechols and carbohydrates, also were detected in the hydrolysates. The proteins became radiolabeled when [1-14C]-beta-alanine was injected into pharate pupae, presumably by the formation of adducts with N-beta-alanyldopamine metabolites during sclerotization. Mild acid hydrolysis released N-beta-alanylnorepinephrine and 3,4-dihydroxyphenylketoethanol from both proteins. Strong acid hydrolysis yielded predominantly 3,4-dihydroxyphenylketoethylamine (arterenone), but also DOPA and dopamine. The N-terminal amino acid sequences of the two cuticular proteins were dissimilar, and that of MSCP246 was more hydrophobic than MSCP120. Both of these proteins were glycosylated with glucose, N-acetylglucosamine and traces of N-acetylgalactosamine, and MSCP246 also contained galactose. These structural glycoproteins, which occur in cuticle undergoing sclerotization, apparently react post-translationally with quinonoid tanning agents to yield catecholamine-protein adducts. Small amounts of peptidyl DOPA probably are formed by hydroxylation of tyrosyl residues. Results from this study are consistent with the hypothesis that these catechol-containing glycoproteins participate in cross-linking reactions in M. sexta pupal cuticle during sclerotization.

Catecholamine-containing proteins from the pharate pupal cuticle of the tobacco hornworm, Manduca sexta.

Three catecholamine-containing proteins from tanning, pharate pupal, abdominal cuticle of the tobacco hornworm, Manduca sexta, have been purified to apparent homogeneity and characterized. These proteins have apparent molecular masses of 32, 41 and 48 kDa and were shown by liquid chromatography-electrochemical analysis, after heating in 1 M acetic acid at 110 degrees C, to release nu-beta-alanylnorepinephrine (NBANE). NBANE is a hydrolysis product of N-beta-alanyldopamine (NBAD) that is bonded covalently at the beta-side-chain carbon to amino acid residues of cuticular proteins. Amino acid compositional analysis revealed that MS-PCP32 (32 KDa) has a high content of alanine (25.6%), valine (13.1%), proline (10.8%) and glycine (10%), a low level of phenylalanine, and no detectable tyrosine or methionine. In contrast, MS-PCP41 (41 kDa) had a much higher content of glycine (31.2%) and substantial levels of serine (9.2%), proline (10.2%) and glutamate/glutamine (10.6%), whereas tyrosine and phenylalanine were not detected. Two of the three purified proteins showed apparent similarities to each other in nu-terminal amino acid sequences, and to several other known insect cuticular proteins. Proteins MS-PCP41 and MS-PCP48 had the characteristic GGX triplet repeat, which is found in a variety of cuticular proteins and may be important for protein folding appropriate for cuticular functions. Therefore, a diversity of cuticular proteins with different amino acid sequences and properties apparently are secreted into the presumptive pupal exocuticle. These then can form adducts and possible cross-links with NBAD through its quinonoid intermediates during cuticular sclerotization.

alpha-Amylase inhibitors from wheat: amino acid sequences and patterns of inhibition of insect and human alpha-amylases.

Four alpha-amylase inhibitors, WRP24, WRP25, WRP26, and WRP27, were purified from wheat flour by preparative, reversed-phase high performance liquid chromatography. All have polypeptide molecular masses of about 14 kDa and are members of the cereal superfamily of protease and alpha-amylase inhibitors. Sedimentation velocity analysis indicated that WRP25 and WRP27 are monomeric proteins, whereas WRP24 is a dimer. WRP24 is identical in N-terminal amino acid sequence to the well characterized 0.19 dimeric inhibitor from wheat kernels. WRP25 and WRP26 differ in sequence from each other at only three positions and represent previously unseparated forms of the 0.28 wheat inhibitor. WRP27 is a previously uncharacterized inhibitor and is more similar in sequence to the 0.28 inhibitor than to the 0.19 inhibitor. WRP25 and WRP26 inhibited alpha-amylases from the rice weevil, red flour beetle, and the yellow meal worm, but did not inhibit human salivary alpha-amylase. WRP24 inhibited the human as well as the insect alpha-amylases, but inhibited one of the two rice weevil alpha-amylases much more strongly than the other. WRP27 was notable in that, of the enzymes tested, it strongly inhibited only the rice weevil alpha-amylases. We observed that the growth rate of red flour beetle larvae was slowed when purified WRP24 was included in the diet at a level of 10%. Addition of WRP24 to corn starch resulted in greater weight loss of red flour beetle adults than occurred on control diets. Our results support the hypothesis that these alpha-amylase inhibitors provide wheat seeds with a selective evolutionary advantage since the inhibitors can slow the growth of insect pests that attack cereal grains.

Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae).

Increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to Tribolium castaneum larvae, and the properties of digestive proteinases were compared in vitro. Cysteine proteinases were the major digestive proteinase class in control larvae, and serine proteinase activity was minor. Dietary serine proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine proteinase inhibitors, there was a dramatic shift from primarily cysteine proteinases to serine proteinases in the proteinase profile of the midgut. Moreover, a combination of cysteine and serine proteinase inhibitors in the diet prevented this shift from cysteine proteinase-based digestion to serine proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.

Anti-adhesive and antibacterial properties of a proprietary denture cleanser.

The antimicrobial efficacy and anti-adhesive attributes of a proprietary denture cleanser were evaluated. To determine cleanser antimicrobial efficacy, Streptococcus mutans was grown on denture acrylic strips which were then exposed to the cleanser. To evaluate anti-adhesive efficacy, the strips were treated with the cleanser and then placed in the Strep. mutans suspension. Following incubation, adhered bacteria were removed and enumerated by viable counting. Treated denture acrylic plates were also placed in a parallel-plate flow chamber and then exposed to Strep. oralis. Images of adhered bacteria were analysed to determine biofilm coverage. Biofilm removal force was quantified by increasing the flow rate between the acrylic plates. The cleanser exhibited a 100% kill against Strep. mutans adhered to the acrylic surface, and inhibited attachment of cells by 66%. The flow chamber study found that cleanser-treated denture acrylic allowed the formation of a multilayer biofilm which was easily removed by a slight increase in flow rate.

An acrylic ampoule represents a denture surface in a microcalorimetric biofilm problem.

An acrylic microcalorimetric ampoule with inserts represented a denture surface in a Streptococcus mutants biofilm study. The biofilm of micro-organisms did not contain as many micro-organisms as those previously studied in planktonic suspension. A more sensitive microcalorimeter (Thermometric's thermal activity monitor) was required to detect the metabolism of S. mutants adhered to an engineered acrylic flow-through ampoule. Biofilm heat outputs were obtained in the acrylic ampoule following standardized procedures with cryopreserved aliquots of a homogeneous suspension of S. mutants.

The effects of surface roughness and type of denture acrylic on biofilm formation by Streptococcus oralis in a constant depth film fermentor.

AIMS: To investigate the effects of surface roughness and type of denture acrylic on the early development of a Streptococcus oralis biofilm in a constant depth film fermentor (CDFF). METHODS AND RESULTS: Streptococcus oralis was incubated with acrylic of known surface roughness in the CDFF. Adherent Strep. oralis were enumerated by viable counting. Cold-cure acrylic was rougher (P < 0.01) than heat-cure acrylic after polishing with abrasive paper of any given grit-grade. Heat-cure acrylic was colonized by fewer (P < 0.001) bacteria than cold-cure acrylic at any given surface roughness. The number of bacteria adhering to heat-cure and cold-cure acrylic increased linearly with mean surface roughness after 2 h incubation, the increase being greater (P < 0.001) for the cold-cure compared with the heat-cure acrylic. However, after 4 h incubation, surface roughness appeared to have no effect on the number of adherent bacteria. CONCLUSION: The type of acrylic used, and its roughness, affect the early stages of biofilm formation by Strep. oralis. SIGNIFICANCE AND IMPACT OF THE STUDY: Choosing an appropriate type of smooth acrylic could lead to reduced biofilm formation in vivo.

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone.

When Manduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both alpha-MDH and alpha-fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 microgram/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.

A microcalorimetric comparison of the anti-Streptococcus mutans efficacy of plant extracts and antimicrobial agents in oral hygiene formulations.

AIMS: This study aimed to evaluate the efficacy of 'natural' putative antimicrobial agents against Streptococcus mutans and to compare these with synthetic agents using the flow microcalorimeter. Streptococcus mutans is one of the oral pathogens responsible for dental caries. METHODS AND RESULTS: Traditional microbiological techniques are invasive and destructive unlike flow microcalorimetry. This rapid technique was used to continuously monitor the power output (bioactivity) of Strep. mutans with reproducibility, precision and accuracy. The antibacterial agents found in oral hygiene products and all the natural agents tested showed anti-Strep. mutans ability. CONCLUSION: In this study microcalorimetry identified agents that had a biological effect and quantified the rate of kill achieved enabling four broad categories of antimicrobial agent to be defined. SIGNIFICANCE AND IMPACT OF THE STUDY: Microcalorimetric data are a better indication of antimicrobial efficacy than merely determining concentrations at which an antimicrobial agent is bacteriostatic or bactericidal.

Comparison of black coral skeleton and insect cuticle by a combination of carbon-13 NMR and chemical analyses.

Cross-polarization, magic-angle spinning 13C NMR spectra of skeletal components of individual colonies of the New Zealand black coral, Antipathes fiordensis, have a marked similarity to spectra of the sclerotized exoskeleton of the adult tobacco hornworm, Manduca sexta. NMR analysis estimates the organic content of the load-bearing skeletal base of A. fiordensis as 70% protein, 10% chitin, 15% diphenol, and 5% lipid by weight, and that of M. Sexta moth cuticle as 60% protein, 20% chitin, 15% diphenol, and 5% lipid. The younger pinnules or tips of A. fiordensis are less than 3% diphenol by weight. The only diphenols extracted from coral skeleton by hydrochloric acid are 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) and 3,4-dihydroxybenzaldehyde (DOBAL), while the predominant diphenols in acid extracts of insect cuticles are N-acyldopamines. More DOPA is found in the base than in the tips of A. fiordensis and it appears to be a peptidyl component of coral skeletal protein. The oxidation of DOPA and DOBAL to quinones may provide mechanical stabilization of the coral skeleton by cross-linking of structural proteins to other proteins or to chitin.

Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.

Characterization of products from the reactions of N-acetyldopamine quinone with N-acetylhistidine.

When insects harden or sclerotize their exoskeletons, quinones of N-acetylated catecholamines such as N-acetyldopamine (NADA) undergo nucleophilic addition reactions with amino acids such as histidine in cuticular proteins. To determine the products that might form when this type of reaction occurs during cuticle sclerotization, the reactions between electrochemically prepared NADA quinone and N-acetylistidine (NAcH), a protein model nucleophile, have been investigated at pH 7. Two major products, 6-[N-(N-acetylhistidyl)]-N-acetyldopamine and 2-[N-(N-acetylhistidyl)]-N-acetyldopamine, were purified by semipreparative reversed-phase liquid chromatography and identified by mass spectrometry and nuclear magnetic resonance spectroscopy. The relative molar ratio of the C(6) mono-addition adduct to the C(2) mono-addition adduct is 87:13. UV/vis spectroscopic analysis shows that both products have an absorption maximum at 284 nm. Cyclic voltammetry shows that these adducts are oxidized less readily than NADA.

1H and 13C NMR of 3-O and 4-O conjugates of dopamine and other catecholamines.

Dopamine and its conjugates are widely distributed among biological species and are utilized for a variety of functions. Insects metabolize dopamine for cuticle melanization and sclerotization. Among the most abundant dopamines found in the larval and pupal development stages of Manduca sexta, the tobacco hornworm, are N-acetyldopamine and N-beta-alanyldopamine. In addition, glycosylated derivatives of these dopamines are found mainly in the hemolymph just prior to cuticulogenesis. The 1H and 13C NMR resonances of dopamine, its 3-O-methyl, 4-O-methyl, N-acetyl, and N-beta-alanyl derivatives, norepinephrine, 4-O-(beta-D-glucuronopyranosyl)dopamine, and the glycosylated products of N-beta-alanyldopamine and dopamine have largely been assigned. Assignments were based on one- and two-dimensional NMR analyses of the above compounds combined with that of specifically enriched [C7-13C]dopamine. 1H NMR showed that the major glycosylated natural product isolated from M. sexta pupal hemolymph was a 3-O-glycosyl derivative of N-beta-alanyldopamine. 13C NMR confirmed that the carbohydrate was D-glucose probably in a beta-linkage. 1H NMR of the aromatic ring protons provided the most definitive method to distinguish 3-O- from 4-O-derivatives of dopamine. In addition, the 3-O-glucosyl conjugate of N-beta-alanyldopamine had unique chemical shifts and coupling patterns compared to those of 4-O-(beta-D-glucuronosyl)- and 3-O-(beta-D-glucopyranosyl)dopamine.

A novel pink-pigmented facultative methylotroph, Methylobacterium thiocyanatum sp. nov., capable of growth on thiocyanate or cyanate as sole nitrogen sources.

The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase -3 micromol NADH oxidized min-1 (mg crude extract protein)-1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [Vmax 634 +/- 24 nmol NH3 formed min-1 (mg protein)-1] than on cyanate [89 +/- 9 nmol NH3 min-1 (mg protein)-1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (Km 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (Ki 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 mM thiosulfate, but unaffected by 0.25 mM sulfide or 1 mM thiosulfate. Polypeptides that might be a cyanase subunit (mol.wt. 17.9 kDa), a cyanate (and/or thiocyanate) permease (mol.wt. 25.1 and 27.2 kDa), and a putative thiocyanate hydrolase (mol.wt. 39.3 kDa) were identified by SDS-PAGE. Correlation of the growth rate of cultures with thiocyanate concentration (both stimulatory and inhibitory) and the kinetics of cyanase activity might indicate that growth on thiocyanate involved the intermediate formation of cyanate, hence requiring cyanase activity. The very high activity of cyanase observed during growth on thiocyanate could be in compensation for the inhibitory effect of thiocyanate on cyanase. Alternatively, thiocyanate may be a nonsubstrate inducer of cyanase, while thiocyanate degradation itself proceeds by a carbonyl sulfide pathway not involving cyanate. A formal description of the new species (DSM 11490) is given.

Insect resistance of transgenic tobacco expressing an insect chitinase gene.

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control.

Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae).

The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut proteinases occurred at pH 4.2. The synthetic substrate N-alpha-benzoyl-DL-arginine-rho-nitroanilide was hydrolyzed by T. castaneum gut proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe rho-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine proteinase inhibitors from potato, Job's tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum proteinases. Other inhibitors of T. castaneum proteinases included leupeptin, L-trans-epoxysuccinylleucylamido [4-guanidino] butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cysteine proteinase inhibitors from potato, Job's tears, or sea anemone. Levels of inhibition were similar to that observed with E-64, although the moles of proteinaceous inhibitor tested were approximately 1000-fold less. These proteinaceous inhibitors are promising candidates for transgenic seed technology to reduce seed damage by T. castaneum.