Effects Of Protease Inhibitors Research Articles

Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta

BackgroundPlants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Plants have developed various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%.ResultsThe aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive responses. We found that larvae fed on tomato transgenic plants co-expressing both proteinase inhibitors showed a notable reduction in weight. Moreover, only 56% of these larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds.ConclusionOur results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.

Effects of proteinase inhibitor from Adenanthera pavonina seeds on short- and long term larval development of Aedes aegypti.

Currently, one of the major global public health concerns is related to the transmission of dengue/yellow fever virus by the vector Aedes aegypti. The most abundant digestive enzymes in Ae. aegypti midgut larvae are trypsin and chymotrypsin. Since protease inhibitors have the capacity to bind to and inhibit the action of insect digestive proteinases, we investigated the short- and long-term effects of Adenanthera pavonina seed proteinase inhibitor (ApTI) on Ae. aegypti larvae, as well as a possible mechanism of adaptation. ApTI had a significant effect on Ae. aegypti larvae exposed to a non-lethal concentration of ApTI during short- and long-duration assays, decreasing survival, weight and proteinase activities of midgut extracts of larvae. The zymographic profile of ApTI demonstrated seven bands; three bands apparently have trypsin-like activity. Moreover, the peritrophic membrane was not disrupted. The enzymes of ApTI-fed larvae were found to be sensitive to ApTI and to have a normal feedback mechanism; also, the larval digestive enzymes were not able to degrade the inhibitor. In addition, ApTI delayed larval development time. Histological studies demonstrated a degeneration of the microvilli of the posterior midgut region epithelium cells, hypertrophy of the gastric caeca cells and an augmented ectoperitrophic space in larvae. Moreover, Ae. aegypti larvae were incapable of overcoming the negative effects of ApTI, indicating that this inhibitor might be used as a promising agent against Ae. aegypti. In addition, molecular modeling and molecular docking studies were also performed in order to construct three-dimensional theoretical models for ApTI, trypsin and chymotrypsin from Ae. aegypti, as well as to predict the possible interactions and affinity values for the complexes ApTI/trypsin and ApTI/chymotrypsin. In this context, this study broadens the base of our understanding about the modes of action of proteinase inhibitors in insects, as well as the way insects adapt to them.

Effects of proteinase inhibitors from Momordica cochinchinensis on several meat-spoilage bacteria

This study aims at primarily investigating the effects of Momordica cochinchinensis proteinase inhibitors (MCoPIs) on several common meat-spoilage bacteria. The obtained results showed that the MCoPIs solution inhibited the growth of 4 studied bacteria: Bacillus cereus, Bacillus cereus 3, Bacillus cereus 81 and Escherichia coli. The proteinase activity (PA) of the bacterial broths supernatants reached the highest level after culturing 48-60 hrs, at 200 rpm and 37oC. The best PA of the studied bacteria was around pH = 7.6. Using PAG containing casein electrophoresis method, several clarified PA bands on dark blue ground were detected from broths supernatants of these bacteria. However, after treatment with MCoPIs solution most of these PA bands were less or disappeared. Treatment of meat with MCoPIs solution showed a positive effect on the freshness of meat. After 16hrs, only the meat samples treated with MCoPIs were remained fresh, while the control samples became rancid. Moreover, the soluble proteins as well as hydrolytic products from samples treated with MCoPIs solution were about 1,5-2 times lower than those of the corresponding control samples. The results indicated an applicable potentiality of MCoPIs solution for protection of meat from bacterial infection and prolonging meat freshness.

Enzymatic Response of the Eucalypt Defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a Bis-Benzamidine Proteinase Inhibitor

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48 h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.

Effect of proteinase inhibitors on Indian alfalfa weevil (Hypera postica Gyll.) growth and development

Three families of proteinase inhibitors, namely, serine, cysteine (thiol) and aspartic (carboxyl) were examined for their inhibitory effects on growth and development of Indian alfalfa weevil (Coleoptera: Curculionidae). Proteinase inhibitors are considered as a part of alternate strategy to control the herbivorous insect as they inhibit the digestive enzymes of the insects. Larval leaf feeding, survival, pupation and adult emergence were significantly decreased by pHMB, (p-hydroxy-mercuribenzoic acid), cystatin and E-64 (trans-epoxysuccinyl-l-leucylamido-(4guanidino)-butane) belonging to cysteine class of proteinases, at a concentration of 0.1 and 0.5%. Serine and aspartic classes of inhibitors have low detrimental effects on larvae. The results demonstrate the inhibitory response of specific proteinase inhibitors on alfalfa weevil larval leaf feeding, survival, pupation and adult emergence. Weevil resistant species, namely, Medicago scutellata showed high level of leaf consumption under forced feeding in vivo bioassay indicated the presence of resistance factors other than proteinase inhibitors.

The effect of proteinase inhibitors in food protein hydrolysis by digestive proteinases of white shrimp (Penaeus vannamei) larvae

AbstractThis study describes the digestive capacities of 10‐day‐old white shrimp postlarvae (PL10) and how some inhibitors of proteinases affect the digestion of protein in aquafeeds by using in vitro hydrolysis techniques. Biochemical data showed eight active proteinases in the PL10 hepatopancreas extract. Enzymes belong to the metallo‐ and serine‐proteinase classes. The effect of inhibitors present in protein ingredients and aquafeeds on PL10 proteinases showed that ovalbumin alone and in commercial microcapsules yielded a significant inhibition in proteolytic activity of PL10 hepatopancreas enzymes. The capacity of PL10 proteinases to hydrolyse the protein fraction within different sources and microcapsules was demonstrated by two in vitro approaches, the pH‐stat (degree of hydrolysis, DH) and electrophoresis (coefficient of protein degradation, CPD). It was shown that PL10 proteinases hydrolyse, in different extent protein sources and microcapsules. Casein, cuttlefish meal and feeds containing these ingredients are quickly hydrolysed. By contrast, ovalbumin and microcapsules containing ovalbumin are not hydrolysed. Copyright © 2006 Society of Chemical Industry

Characterization of serine proteases in the monogenean Neobenedenia girellae

Proteases of Neobenedenia girellae were examined by using zymographic analysis. Zymography of N. girellae homogenate revealed proteases at approximately 88, 107, 149 and 167 kDa for the adult worms and approximately 147 and 166 kDa for the oncomiracidia, respectively. The enzyme activities were inhibited by Pefabloc SC but were not inhibited by E-64 and Pepstatin A, indicating that these proteases were serine proteases. The gelatinolytic activities of these proteases were more active at pH 8.5, when pH range of 5.5–11.5 was examined, and increased with the increase of temperature within the range of 15–37 °C. Further, the effect of proteinase inhibitors, pHs and temperatures on N. girellae were examined. Egg laying of mature N. girellae was suppressed by Pefabloc SC and seawater of pH 5.5 to 6.5 and at pH 9.5. Additionally, egg laying of mature N. girellae was suppressed by low incubation temperatures (10, 15, 20 °C). The oncomiracidia treated with Pefabloc SC and seawater of pH 5.5 and pH 9.5 were also affected and cilia of the treated parasites ceased moving. These results suggest that serine proteases of the parasite could be important for their survival, and their physiological active substances such as proteases, may have adapted to higher water temperature and tend to be suppressed at pH 5.5–6.5 and pH 9.5. The results of this study could be informative to develop an effective control method against outbreaks of the parasites.

Effects of proteinase inhibitors on fertilization in sea lamprey ( Petromyzon marinus)

A search for alternative sterilants in parasitic fish encouraged us to explore the usefulness of proteinase inhibitors for this purpose. Fertilization in sea lamprey species ( Petromyzon marinus L.) was inhibited by chymotrypsin and trypsin inhibitors 4′-acetamidophenyl 4-guanidinobenzoate (AGB), chymostatin, tosyl- l-lysine chloromethyl ketone (TLCK), and N-tosyl- l-phenylalanine chloromethyl ketone (TPCK) when these substances were added into a fertilization medium at the time of fertilization. Preincubation of eggs before fertilization with 100 μM TPCK, but not TLCK, resulted in inhibition of fertilization. Conversely, preincubation of spermatozoa with TLCK, but not TPCK, produced inhibition of fertilization. These data suggest the involvement of the chymotrypsin-like activity of eggs and trypsin-like activity of spermatozoa in fertilization. However, enzymes present in sperm suspensions were able to hydrolyze a chymotrypsin substrate N-glutaryl- l-phenylalanine- p-nitroanilide (GPNA) but not trypsin substrate N-α-benzoyl- dl-arginine- p-nitroanilide (BAPNA). The nature of this activity can be characterized as serine protease and our results indicate the involvement of serine proteinases in the fertilization of sea lamprey.

The effect of proteinase inhibitors on glomerular albumin permeability induced in vitro by serum from patients with idiopathic focal segmental glomerulosclerosis.

The putative circulating factor responsible for the glomerular permeability alterations induced in vitro by serum from patients affected by focal segmental glomerulosclerosis (FSGS) remains unidentified. We have observed that a serine proteinase isolated from patient serum increases albumin permeability in isolated glomeruli. The objective of the present study was to determine the effect of various proteinase inhibitors on glomerular albumin permeability (P(alb)) in isolated glomeruli incubated with FSGS serum. The study population consisted of 12 FSGS patients (eight males; mean age: 21+/-10 years) previously shown to have elevated serum albumin permeability activity. P(alb) was determined by measuring the change in glomerular volume induced by applying oncotic gradients to isolated healthy rat glomeruli treated with patient serum in comparison to control serum. Solutions of seven different proteinase inhibitors (0.5 mg/ml) were added to the incubation media with the sera (1:1 vol/vol): serine proteinase inhibitors (PMSF, leupeptin, aprotinin, gabexate mesylate), the cysteine proteinase inhibitor E-64, the metalloproteinase inhibitor EDTA and the aspartate proteinase inhibitor pepstatin. Sera from the same patients were also tested with the addition to the incubation media of quinaprilat, an inhibitor of the metalloproteinase angiotensin-converting enzyme. Mean P(alb) of the sera was 0.86+/-0.11, with the addition of PMSF 0.41+/-0.09, leupeptin 0.30+/-0.17, aprotinin 0.09+/-0.14, gabexate mesylate 0.27+/-0.25, E-64 0.81+/-0.09, EDTA 0.68+/-0.10 or pepstatin 0.76+/-0.11. The mean P(alb) of the sera combined with quinaprilat was reduced to 0.34+/-0.35. Thus, only the serine proteinase inhibitors consistently blocked the increased P(alb) induced by the FSGS sera. In the cascade of events that lead to the initiation of glomerular fibrosis in FSGS, the putative glomerular permeability factor associated with FSGS may require a serine proteinase to effect its activity.

Cowpea ( Vigna unguiculata) vicilins bind to the peritrophic membrane of larval sugarcane stalk borer ( Diatraea saccharalis)

In this work, we show that vicilins from two Vigna unguiculata (cowpea) genotypes, Epace-10 and IT 81D-1045, which are susceptible and resistant to attack by the cowpea weevil Callosobruchus maculatus, respectively, associate with the peritrophic membrane (PM) from larvae of Diatraea saccharalis. Solutions with increasing concentrations of vicilins were incubated with PM of the larvae and subsequently analysed by electrophoresis with SDS. It was observed that the majority of the bands of approximately 50,000 Da (characteristic of vicilins) did not appear in the separating gel and only lower molecular weight polypeptides were seen. When vicilins were incubated with PM, and the solution was then heated after the incubation, the band pattern in the gel appeared completely different. It was observed that the vicilins were being hydrolysed by proteinases associated with the PM. When the incubated samples were heated after the reaction, the major bands reappeared, demonstrating that most of the vicilin molecules had bound to the PM of D. saccharalis. These results suggest that when the vicilins are in contact with the PM they are bound and also digested by the PM of this insect. The major and several minor proteinases from the PM were extracted with Triton X-100 and their activity and the inhibition of this activity were analysed by in gel assays. Based on the effects of proteinase inhibitors, the PM-associated activity is due to serine class proteinases. Larvae of D. saccharalis were fed on artificial diets containing purified vicilins from Epace-10 or IT 81D-1045 seeds. Vicilins from Epace-10 did not affect the larval development, while IT 81D-1045 vicilins reduced significantly the survival rate of the sugar cane borer.

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-α-benzoyl- dl-arginine-ρ-nitroanilide was hydrolyzed by T. castaneum gut proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe ρ-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.

Possible implication of metalloproteinases in post-mortem tenderization of fish muscle

It is suspected that the proteolytic breakdown of extracellular matrix proteins is responsible for the postmortem tenderization of fish muscle during chilled storage. In order to identify the type (s) of proteinases involved in this phenomenon, the effect of proteinase inhibitors, EDTA (ethylenediamine tetraacetic acid), 1, 10-phenanthroline, ρ-APMSF [(ρ-amidinophenyl) methanesulfonyl fluoride hydrochloride] and E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidinobutane)] on tenderization was investigated by using Japanese flounder. Proteinase inhibitor solution was injected into a blood vessel in a caudal portion of live flounder and the firmness of muscle was then evaluated as a shear force value at 0 h and 6 h after death. Metalloproteinase inhibitors, EDTA and 1, 10-phenanthroline, significantly suppressed postmortem tenderization. These findings suggest that metalloproteinases are candidates for proteinases involved in the postmortem tenderization of fish muscles. Although not significantly, ρ-APMSF, a serine proteinase inhibitor, partially suppressed muscle tenderization, which suggests that serine proteinases are also implicated in postmortem tenderization. A cysteine proteinase inhibitor, E-64, showed no effect, suggesting that cysteine proteinases are not involved.

Proteolysis and utilization of albumin by enrichment cultures of subgingival microbiota.

Subgingival dental plaque consists mainly of microorganisms that derive their energy from amino acid fermentation. Their nutrient requirements are met by the subgingival proteolytic system, which includes proteases from microorganism and inflammatory cells, and substrate proteins from sulcus exudate, including albumin. To determine the selective effect of individual proteins on microbiota, we used albumin as the main substrate for growth. Eight subgingval plaque samples from untreated periodontal pockets of patients with adult periodontitis were inoculated in peptone yeast medium with bovine albumin (9 g/l). After three subculture steps, cell yields of the enrichment cultures at the medium with 0, 1.25, 2.5, 5, 10, and 20 g/l albumin were determined. Proteolytic activity (U/absorbance at 550 nm) of the enrichment cultures and different isolates derived from the cultures was estimated by the degradation of resorufin-labeled casein. It was observed that the yield of the mixed culture was albumin limited, and the proteolytic activities of the cultures in albumin broth were higher than in control (peptone broth). Among the isolates from the enrichment cultures, Peptostreptococcus micros, Prevotella melaninogenica, Prevotella buccae and Prevotella bivia demonstrated proteolysis. The frequent occurrence of Streptococcus gordonii and Streptococcus anginosus in the albumin cultures is explained by their ability to utilize arginine as an energy source for growth. Albumin in the medium was partly degraded by pure cultures but completely consumed in enrichment cultures, indicating synergy of bacterial proteinases. It is concluded that the subgingival microbiota possesses proteolytic activity and may use albumin as a substrate for their growth. Enrichment cultures on albumin may serve as a relatively simple in vitro model to evaluate the effects of proteinase inhibitors.

Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration.

The infective larvae of Necator americanus were shown to secrete all mechanistic classes of proteolytic enzymes with two overall pH optima of 6.5 and 8.5 using fluorescein isothiocyanate-labeled casein as the substrate. Since infective larvae are obligate skin penetrators, the effect of each of these enzyme classes against macromolecules derived from human skin was examined. Larval secretions were shown to degrade collagen types I, III, IV, and V, fibronectin, laminin, and elastin. All the skin macromolecules tested were hydrolyzed by aspartyl proteinase activity, which was inhibitable by pepstatin A. Collagen and elastin was also hydrolyzed by metalloproteinase activity, while the serine proteinase activity hydrolyzed only elastin. As a consequence of these experiments, the effect of proteinase inhibitors on the penetration of live larvae through hamster skin was tested. Larval penetration was significantly inhibited only by pepstatin A, confirming the importance of the aspartyl proteinase activity during the skin penetration process.

Characterization of digestive proteases in the weevil Aubeonymus mariaefranciscae and effects of proteinase inhibitors on larval development and survival

AbstractThe major digestive proteinase activities of a new sugar beet pest, Aubeonymus mariaefranciscae Roudier (Coleoptera: Curculionidae), were characterized. Both larvae and adults of A. mariaefranciscae were found to use a complex proteolytic system for protein digestion based on at least trypsin‐, chymotrypsin‐, elastase‐, cathepsin D, leucine aminopeptidase‐, carboxypeptidase A‐ and carboxypeptidase B‐like activities. An azocaseinolytic activity at pH 5.0–7.0 was identified, that was not affected by specific inhibitors and activators, making its classification in any of the mechanistic classes established not possible. According to this proteolytic profile, several serine proteinase inhibitors were tested in vitro and in vivo to establish their potential as resistance factors against A. mariaefranciscae. Larvae fed from neonate to pupation on diets containing 0.2% (w/w) soybean Bowman‐Birk trypsin‐chymotrypsin inhibitor, soybean Kunitz trypsin inhibitor, turkey egg white trypsin inhibitor, or lima bean trypsin inhibitor endure lower survival rates and display significant delays in the developmental time to pupation and to adult emergence. Interestingly, the most significant levels of mortality (about 90%) occurred with larvae fed on diets containing a combination of two or three inhibitors, suggesting a synergistic toxicity.

The effect of proteinase inhibitors (PI) on the incidence of oropharyngeal candidiasis (OPC) in an HIV cohort

The effect of proteinase inhibitors (PI) on the incidence of oropharyngeal candidiasis (OPC) in an HIV cohort

O-53 - Effects of proteinase inhibitors and Bcl-2 on 6-OHDA-induced cell death of neuronal PC 12 cells and primary cultured microglia

O-53 - Effects of proteinase inhibitors and Bcl-2 on 6-OHDA-induced cell death of neuronal PC 12 cells and primary cultured microglia

The role of cuticle-degrading proteases in fungal pathogenesis of insects

The proteinaceous outer integument of insects forms an effective barrier against most microbes. Only the 700 known species of entomopathogenic fungi effect entry into their hosts by breaching the cuticle. There is accumulating evidence that the ability of fungi to degrade protein may aid their invasion of and growth in this orderly complex structure. Evidence for the particular importance of proteinases derives largely from studies of their production in infected cuticles associated with cuticle degradation, the effects of proteinase inhibitors on pathogen behavior, and by the analysis of protease-deficient mutants. More recently, studies have included the cloning, identification, and manipulation of specific protease genes of Metarhizium anisopliae, particularly those of the subtilisin (chymoelastase) type (designated Pr1) also produced by many other entomopathogenic fungi. Following solubilization of cuticle proteins by Pr1-type endoproteases, complete degradation of the cuticle involves a number of interacting enzyme species including a family of trypsin-like proteinases, metalloproteinases, several aminopeptidases, and a carboxypeptidase. Testing genetically engineered M. anisopliae null mutants of Pr1 indicated that the other endopeptidases can partially substitute for Pr1. The exopeptidases further degrade peptides released by the endopeptidases producing free amino acids for uptake and metabolism. Utilization of these enzymes has assisted investigators in understanding cuticle structure and how the cuticle is degraded naturally, and could lead to improved strain selection of entomopathogenic fungi or the introduction of their genes into other microbes and plants for the purpose of insect control. Key words: proteinaceous insect cuticle, pathogen endopeptidases, exopeptidases, multiple isozymes, enzyme regulation.

Selected Proteinase Inhibitor Effects on Alfalfa Weevil (Coleoptera: Curculionidae) Growth and Development

Proteinase inhibitors, which can inhibit digestive enzymes of specific insects, are a potential alternate control mechanism against herbivorous insects. An in vivo laboratory bioassay was developed to screen proteinase inhibitors for their ability to inhibit growth and development of the alfalfa weevil, Hypera postica (Gyllenhal). Larval foliar feeding, pupation, and adult emergence were significantly decreased by E-64, pHMB, and leupeptin, inhibitors of cysteine proteinases, at a concentration of 0.1%. Aspartic proteinase inhibitors (antipain and pepstatin) demonstrated mixed results at 0.1 and 0.5% concentrations. Serine proteinase (trypsin) inhibitors had no effect on any of the variables measured at 0.5%. These observations suggest that alfalfa weevil larvae use cysteine proteinases as major digestive enzymes and that other proteinases may be present as well. In vivo studies on the effects of proteinase inhibitors on herbivorous insects, in most cases, have been determined by measuring larval development (weight) and mortality. This study reports on the negative effects of specific proteinase inhibitors on insect larval foliar feeding, pupation, and adult emergence when ingested with an insect℉s preferred host.

Collagen degradation within subcutaneous air pouches in vivo: The effects of proteinase inhibitors

A straightforward in vivo model of collagen degradation is described that can be used to measure the effects of different classes of proteinase inhibitors. Air pouches, formed subcutaneously in the dorsal thoracic region of rats, were inflamed 6 to 8 days later by injecting λ-type carrageenan. 14C-Collagen was injected into the air pouches either 1 day before or 1 day after λ-carrageenan-induced inflammation: in the latter case, the inflammatory exudate fluid was drained from the air pouches immediately prior to administering 14C-collagen. Ninety percent of the 14C-collagen was degraded and cleared within 3 days from pre-inflamed air pouches, but degradation was much slower from the post-inflamed or non-inflamed air pouches. Proteinase inhibitors injected simultaneously with the 14C-collagen, and again 6 hr later, reduced the extent of 14C-collagen degradation from air pouches measured after 24 hr. Forty-two percent of the degradation of 14C-collagen could be inhibited by a mixture of enzyme inhibitors (leupeptin, α 1-anti-proteinase, aprotinin, and pepstatin) injected together with 1,10 phenanthroline, the zinc metalloenzyme inhibitor. The 1,10 phenanthroline alone caused a 33% inhibition of 14C-collagen degradation, and the inhibitor mixture given alone inhibited 14C-collagen loss by 25%. Approximately 60% of the degradation of 14C-collagen in this model was mediated by mechanisms resistant to this combination of proteinase inhibitors, which may indicate the significant involvement of non-enzymic modalities, or degradation in intracellular compartments inaccessible to extracellular agents.