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Associational effects, whereby plants influence the biotic interactions of their neighbors, are an important component of plant–insect interactions. Plant chemistry has been hypothesized to mediate these interactions. The role of chemistry in associational effects, however, has been unclear in part because the diversity of plant chemistry makes it difficult to tease apart the importance and roles of particular classes of compounds. We examined the chemical ecology of associational effects using backcross‐bred plants of the Solanum pennellii introgression lines. We used eight genotypes from the introgression line system to establish 14 unique neighborhood treatments that maximized differences in acyl sugars, proteinase inhibitor, and terpene chemical diversity. We found that the chemical traits of the neighboring plant, rather than simply the number of introgression lines within a neighborhood, influenced insect abundance on focal plants. Furthermore, within‐chemical class diversity had contrasting effects on herbivore and predator abundances, and depended on the frequency of neighboring plant chemotypes. Notably, we found insect mobility—flying versus crawling—played a key role in insect response to phytochemistry. We highlight that the frequency and chemical phenotype of plant neighbors underlie associational effects and suggest this may be an important mechanism in maintaining intraspecific phytochemical variation within plant populations. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality. Therefore, the mining of V. dahlia-resistance genes is urgently needed. Proteases and protease inhibitors play crucial roles in plant defense responses. However, the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown. This study provides a comprehensive analysis of the PR6 gene family in the cotton genome. We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family, which belongs to the potato protease inhibitor I family of inhibitors. Thirty-nine PR6s were identified in Gossypium arboreum, G. raimondii, G. barbadense, and G. hirsutum, and they were clustered into four groups. Based on the analysis of pathogen-induced and Ghlmm transcriptome data, GhPR6-5b was identified as the key gene for V. dahliae resistance. Virus-induced gene silencing experiments revealed that cotton was more sensitive to V. dahliae V991 after PR6-5b silencing. The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C). Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V. dahliae, and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V. dahliae.

The effect of silver nanoparticles (Ag NPs) at oral administration on the gastric mucosa of intact animals and possible prevention of acute gastric ulcer formation has been studied. The indices of nonspecific proteinases and their inhibitors in blood serum and the supernatant of gastric mucosal homogenate were evaluated. It has been established that the use of Ag NPs solution as a drinking substrate in intact animals is accompanied by a minimal reaction of the components of proteinase inhibitory systems both at the systemic and local levels. Prophylactic use of Ag NPs solution in modeling acute gastric ulcers leads to inhibition of activation of nonspecific proteinases and preservation of inhibitory potential, which may indicate the presence of anti-inflammatory effects of Ag NPs. The inhibition of the activation of proteolytic enzymes and the preservation of a sufficiently high inhibitory potential of the Ag NPs solution as a preventive means for the formation of an ulcerative defect of the gastric mucosa indicates the presence of anti-inflammatory effects of Ag NPs, which is the basis for further research. [ABSTRACT FROM AUTHOR]

The purpose of the study was to determine the clinical efficiency of proteinase inhibitor usage in patients with burns. Materials and Methods. The results of studies performed in 32 patients with superficial and deep burns were analyzed: 12 patients of the main group, whom were used the proteinase inhibitor drug in the complex treatment, and 20 comparison groups, who were treated according to the standard scheme. The content of calpains, α-1 proteinase inhibitor (α-1-IP), α-2 macroglobulin (α-2-MG) and tumor necrosis factor α (TNF-α) in patients’ peripheral blood were determined. Results. The decrease in proteolytic activity of blood in patients of the main group was determined as a decrease in calpain levels by 1.16 times (50.51 ± 10.32 μEq/min on the 10th-16th day compared to baseline 58.83 ± 3.62 μEq/min on the 1-4th day after injury), while in the comparison group indicators of calpain content did not tend to decrease. Comparing the TNF-α values ​​of the main group and the comparison group, it was found that the values ​​of the main group were significantly lower 1.15 times on the 5th-9th day (245.33 ± 10.36 pg/ml and 281.2 ± 13, 67 pg/ml) and 1.08 times lower on the 10th-16th day (236.60 ± 8.78 pg/ml and 256.35 ± 15.70 pg/ml). The usage of ulinastatin led to a less pronounced decreasing in proteinase inhibitors levels: α-1-IP (79.31 ± 1.54 μmol/l of the main group compared with 72.1 ± 7.8 μmol/l of the control group on the 5th-9th day after injury and 75.34 ± 5.13 μmol/l of the main group compared with 68.0 ± 4.9 μmol/l of the control group on the 10-16th day after injury), α-2-MG (2.63 ± 0.24 g/l of the main group compared with 2.2 ± 0.4 g/l of the control group on the 10-16th day after injury). Conclusion. The results of the study indicate that the proteinase inhibitor drug included in the standard treatment regimen for acute burns optimizes the proteolytic activity of peripheral blood, promotes the inflammatory response optimal development, inhibits the endothelial dysfunction development.

Profiling of proteinase inhibitors (PIs) was done on the diverse group of sugarcane viz., exotic clones, hybrids and Erianthus arundinaceus. Their inhibitory activity against commercial trypsin enzyme and midgut protease of sugarcane borers was investigated. Proteinase inhibitors were extracted from leaf sheath, meristem and stalk tissue of selected groups. The results revealed that the PIs were in maximum quantity in meristem followed by leaf sheath and stalk tissue of hybrids and E. arundinaceus. The trypsin inhibition was maximum in leaf sheath (11.51- 32.26%), meristem (15.86- 90.94%) and stalk tissue (1.62- 39.62%) of E. arundinaceus compared to those of hybrids and exotic clones. Subsequently, extracted meristem and stalk tissue PIs were assayed against midgut protease of sugarcane early shoot borer (ESB) and internode borer (INB). The results showed that PIs from E. arundinaceus meristem significantly inhibited the midgut protease of ESB and INB up to >70 and >60%, respectively. However, the PI from the meristem of exotic clones and hybrids showed considerable inhibition to midgut proteases of ESB. The PIs from stalk tissue of all groups were ineffective as regards gut protease inhibition in the sugarcane borers. [ABSTRACT FROM AUTHOR]

Currently, little is known about inhibitory substances enabling tapeworms to settle in fish intestines thereby avoiding proteolysis. Contrary to previous studies with certain host–parasite pairs, this research compares the inhibitory capacities in three tapeworm species of the same genus Proteocephalus from four different fishes (P. torulosus from dace and zope, P. sagittus from stone loach and P. cernuae from ruffe). The tapeworm extracts studied significantly reduced the activity of commercial trypsin (although to a lesser degree than the synthetic inhibitor of serine proteinases PMSF), displaying clear inter‐specific variation in worms' inhibitory ability. We also measured the proteolytic activity of the host intestinal mucosa exposed to tapeworm extracts which served as inhibitors. Based on per cent inhibition values, all tapeworm extracts significantly suppressed the mucosal proteolytic activity, with marked differences between certain host–parasite pairs. SDS‐PAGE electrophoresis of the incubation media and extracts detected in each tapeworm species 20–36 protein bands with apparent molecular weights from 10–12 to 312.5 kDa, mostly below 50 kDa. The incubation medium and extract of each parasite shared one to six bands ranging from 12 to 35 kDa, depending on its species, with only four bands common for two or more species. The band profiles suggest that in various Proteocephalus species inhibitory capacities against host proteinases can be ensured by different proteins. [ABSTRACT FROM AUTHOR]

Pigeonpea is an agriculturally important leguminous crop with high vulnerability to insect pest attack specifically, Helicoverpa armigera. The proteinase inhibitors (PIs) mediated host plant resistance against insect pests is a promising sustainable agricultural research practice. The current study was carried out to perceive biochemical characterization of proteinase inhibitors named PPTI in the pigeonpea (cv. BSMR 736). The purification of PPTI from crude protein seed extract was achieved by acetone precipitation, N-LP-IEF, and trypsin affinity chromatography. It was found to inhibit bovine trypsin and HaGPs in vitro. The optimal conditions for inhibition were pH 8 and temperature 40°C. The PPTI showed four isoinhibitors bands on native, non-reducing and reducing SDS-PAGE in the range of 26.7-19.3 KDa. On resolution on two-dimensional gel electrophoresis (2-DE), PPTI produced nine pI variant spots having isoelectric point (pI) 6.6, 6.6, 6.3, 6.1, 5.9, 5.8, 5.7, 5.6, and 5.6. An artificial diet containing PPTI reduced the H. armigera larval weight about 69%, with 25% mortality. For eco-friendly sustainable agricultural practices, natural compounds like PPTI could be expressed in transgenic crops to prevent the invasion of H. armigera in pigeonpea. [ABSTRACT FROM AUTHOR]

Abstract Background Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. Results We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. Conclusions We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X‐100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host–parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths. [ABSTRACT FROM AUTHOR]

Background: Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. Results: We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. Conclusions: We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated. [ABSTRACT FROM AUTHOR]

Breast cancer (BCa) is the leading cause of cancer-related deaths among women. Numerous efforts are being directed toward identifying novel tissue and/or circulating molecular markers that may help clinicians in detecting early-stage BCa patients and in providing an accurate estimation of the prognosis and prediction of response to clinical treatments. In this setting, emerging evidence has indicated Cystatin C (Cyst C), as the most potent endogenous inhibitor of cysteine cathepsins, as a possible useful marker in the clinical management of BCa patients. This review analyzes the results of emerging studies underpinning a potential clinical role of Cyst C, as additional marker in BCa. Cyst C expression levels have been reported to be altered in tumor tissues and/or in biological fluids of BCa patients. Furthermore, clinical evidence has highlighted a significant correlation between altered Cyst C levels in tumor tissues and/or biological fluids and some clinco-biological parameters of BCa progression. These findings provide evidence for a potential clinical use of Cyst C as a novel marker to improve the clinical and therapeutic management of BCa patients and as a gauge for better clarifying the role of cysteine proteinases in the various steps of BCa progression. [ABSTRACT FROM AUTHOR]

Abstract Background Plants 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%. Results The 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. Conclusion Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.

This study demonstrates that soybean cultivars respond differentially to damage in a herbivore-specific manner, and trigger responses decreasing herbivore performance. Soybean crops are affected by a great number of insect herbivores, resulting in devastating yield losses. Secondary metabolites like proteinase inhibitors and phenolic compounds are part of plants' defense mechanisms against insect pests. However, the specificity of soybean defense responses to different attacking herbivores is poorly known. To investigate species-specific foliage responses to herbivory, we used two different commercial soybean cultivars (DM 4210 and DM 5.8i) widely used in Argentina with diverse susceptibility to insects' attack and two species of phytophagous insects, thrips (Caliothrips phaseoli; Thysanoptera) and lepidopteran larvae (Spodoptera frugiperda; Lepidoptera) with a different way of feeding. Benzoic acid derivative levels were increased by damage of both insect species in the foliage of the field-grown soybean cultivars, whereas trypsin protease inhibitors activity was induced in cultivar (cv) DM 4210 by fall armyworm damage and malonyl genistein content in cv DM 5.8i after thrips' attack. Although survivorship and mass of fall armyworm larvae were not differentially affected in field conditions by soybean cultivars, larvae reared in the laboratory that fed on cv DM 5.8i gained more mass than those on DM 4210. Conversely, thrips performance, natural colonization, and preference of feeding were higher on cv DM 4210 than on cv DM 5.8i. The differential effects of soybean cultivars on insectspecies' performance were explained not only by induced defenses, but also by differences of constitutive defenses between cultivars. This study demonstrated that soybean cultivars responded differentially to damage in a herbivore-specific manner, and triggered responses decreased herbivore performance. [ABSTRACT FROM AUTHOR]

The proteolytic activity in washings off the Triaenophorus nodulosus cestode tegument and the ability of the worms to inactivate proteolytic enzymes were studied. It was found that the major proteolytic activity in the washing samples is represented by the easily desorbed fraction most probably characterizing the activity of the host’s enzymes. Serine proteinases are an essential part of these enzymes. It was shown that the worms’ incubation medium and their homogenates can inhibit host proteinases and commercial trypsin samples. Suppressive activity of the incubation medium suggests that the inhibitors are rather spontaneously produced by the worms than induced by the presence of proteinases in the surrounding medium. The inhibitor produced by the cestode is hypothesized to be trypsin-specific.

Adaptive mechanisms underlying the long-term existence of intestinal parasites in their enzymatically hostile environment are still poorly understood, particularly with regard to fish cestodes. The study describes the activity distribution of proteolytic enzymes along the gut of the bream Abramis brama infected with intestinal cestodes Caryophyllaeus laticeps and characterizes the capacity of these worms to inhibit host proteinase activity. Mucosal proteolytic activity was mainly presented by serine proteinases. The research revealed an insignificant increase in total proteolytic activity from anterior and middle to posterior part of the gut accompanied with changes in proportions of various proteinase subclasses along the intestine. The trypsin (but not chymotrypsin) activity in the posterior section was significantly higher than in the mid-section. Both the incubation medium of the worms and their extract had a significant inhibitory effect on mucosal proteolytic activity and commercial trypsin samples. In both instances, the effect was comparable with that of a synthetic serine protease inhibitor, PMSF. SDS-PAGE electrophoregrams of the incubation medium of C. laticeps and its extract revealed three common protein bands, with apparent molecular masses from 19 to 47 kDa, possibly responsible for the worms' inhibitory capacities. According to casein-zymography performed, the target host proteinases for a putative cestode inhibitor (inhibitors) have an approximate molecular weight of 28–53 kDa. A comparative test with the extracts from three other cestodes showed that each of them can suppress the proteolytic activity of the bream mucosa. The level of inhibitory activity was found to increase with protein content in the extracts of these tapeworms. Unlabelled Image • The proteolytic activity of the bream's intestinal mucosa tends to increase posteriorly along the gut. • The incubation medium and extract of C. laticeps significantly suppress the mucosal proteolytic and trypsin activities. • The phoregrams of the incubation medium and extract display three common protein bands likely responsible for inhibition. • Extracts of cestodes from non-host fishes are also able to lower the proteolytic activity of the bream intestinal mucosa. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors with the ability to inhibit specific proteinases are usually closely connected with the immune system. Interestingly, proteinase inhibitors are also a common ingredient in the organic matrix of mollusk shells. However, the molecular mechanism that underlies the role of proteinase inhibitors in immune system and shell mineralization is poorly known. In this study, a Kunitz serine proteinase inhibitor (HcKuPI) was isolated from the mussel Hyriopsis cumingii. HcKuPI was specifically expressed in dorsal epithelial cells of the mantle pallium and HcKuPI dsRNA injection caused an irregular surface and disordered deposition on the aragonite tablets of the nacreous layer. These results indicated that HcKuPI plays a vital role in shell nacreous layer biomineralization. Moreover, the expression pattern of HcKuPI during LPS challenge and pearl formation indicated its involvement in the antimicrobial process during pearl sac formation and nacre tablets accumulation during pearl formation. In the in vitro calcium carbonate crystallization assay, the addition of GST-HcKuPI increased the precipitation rate of calcium carbonate and induced the crystal overgrowth of calcium carbonate. Taken together, these results indicate that HcKuPI is involved in antimicrobial process during pearl formation, and participates in calcium carbonate deposition acceleration and morphological regulation of the crystals during nacreous layer formation. These findings extend our knowledge of the role of proteinase inhibitors in immune system and shell biomineralization. • HcKuPI is involved in antimicrobial process during pearl sac formation. • HcKuPI is specifically expressed in dorsal epithelial cells of the mantle pallium. • HcKuPI dsRNA injection caused disordered aragonite tablets deposition of the nacreous layer. • GST-HcKuPI could increase precipitation and induce overgrowth of calcium carbonate. [ABSTRACT FROM AUTHOR]

A sugarcane wild relative Erianthus arundinaceus was identified as a source of resistance against Chilo infuscatellus, a major devastating pest at the early cropping stage of sugarcane. In the view of earlier studies, inhibitory activities of proteinase inhibitors (PIs) from C. infuscatellus field resistant E. arundinaceus genotypes viz., IK 76 78, IJ 76 400, IK 76 84, IK 76 88, IJ 76 370, FIJI 55, IJ 76 364 and ERI 2798 and a popular variety Co 86032 were done. Although proteinase inhibitors differed significantly among the plant parts of selected genotypes, it was comparatively higher in the apical meristem (70%) followed by leaf sheath (20%) and stalk tissues (11%). The results also showed that E. arundinaceus apical meristem PIs significantly inhibited (> 70%) the midgut proteinase of early shoot borer. Among the genotypes, IJ 76 370 and IJ 76 364 had considerably higher PIs in apical meristem and leaf sheath which showed strong inhibitory activity against gut proteinase of sugarcane shoot borer. Developmental characteristics of the borer showed a significant reduction of larval and pupal survival along with extended larval duration in the genotypes IJ 76 370 and IJ 76 364. Correlation studies indicated negative relationship with shoot borer larval (P < 0.05, r = −0.14) and pupal survival (P < 0.05, r = −0.56**) with apical meristem PIs of E. arundinaceus. Similarly, significant correlations were found between the shoot borer developmental period and apical meristem PIs of E. arundinaceus (P < 0.05, r = 0.52**, P < 0.05, r = 0.24 and P < 0.05, r = 0.56** for larval, pupal and total developmental period of the borer respectively). The study identified two elite genotypes IJ 76 370 and IJ 76 364 which could be used as donors for sugarcane pre-breeding programme for the development of shoot borer resistant varieties. Besides, it could be possible to identify novel insecticidal proteins (PIs) that may be utilized in genetic manipulation work for producing transgenics imparting resistance against sugarcane C. infuscatellus. [ABSTRACT FROM AUTHOR]

The extract of the fish tapeworm Eubothrium rugosum was found to inhibit activity of intestinal proteases in its host, the burbot. A decrease in proteolytic activity of the intestinal mucosa, when using the E. rugosum extract as an inhibitor, is comparable with that of a serine protease inhibitor PMSF. This implies that the tapeworm-produced inhibitor inactivates major intestinal serine proteases, trypsin and chymotrypsin. The tapeworm extract was also demonstrated to inhibit proteolytic activity in the intestinal mucosa of the two other fish species, zope and bream. Using the Gini index, it was found that the distribution of percent inhibition levels shows some nonuniformity across the given set of species. Namely, the tapeworm extract exerts a more selective inhibitory effect on intestinal proteases in different fish species as compared to PMSF. This indicates a partial though quite explicit species-specific selectivity of the tapeworm-produced inhibitor toward the host's proteolytic activity which may contribute to the formation of specific host-parasite interrelationships. [ABSTRACT FROM AUTHOR]

Cystatin C (Cyst C) is an endogenous inhibitor of lysosomal cysteine proteinases, which has been shown to play a role in several normal and pathological processes. Interestingly, a growing number of experimental and clinical studies suggest that this inhibitor also appears to be implicated in the malignant progression of various human tumors. However, the role of Cyst C in malignant diseases is still controversial as these studies have highlighted that this protein may function either as tumor suppressor or tumor promoter. The specific mechanisms underlying these opposing effects at present remain murky and are the subject of many current investigations. On the other hand, a complete knowledge of these mechanisms is of clinical interest in order to develop new, effective antitumor treatments based on the appropriate use of natural and/or synthetic cysteine proteinase inhibitors. This paper discusses the current findings regarding the role of Cyst C in cancer progression and the clinical implications emerging from these studies. [ABSTRACT FROM AUTHOR]

We investigated the effect of presowing treatment of seeds with salicylic (SA) and jasmonic (JA) acids on the growth of wheat Triticum aestivum L. seedlings, generation of hydrogen peroxide (Н2О2) therein, and transcriptional activity of the genes encoding defense proteins—oxalate oxidase (OxO), peroxidase (PO), and proteinase inhibitor (PI)—upon their inoculation with stinking smut pathogen Tilletia caries (DC.) Tull. SA and JA were found to reduce adverse effect of T. caries on growth of seedlings and the extent of their infection by the pathogen. Improvement of plant resistance to T. caries depended on a stimulatory effect of SA and JA on the formation of Н2О2 in plant tissues and changes in activity of ОхО, PO, and catalase. SA was shown to elevate transcriptional activity of the genes of oxalate oxidase and peroxidase. We detected a considerable stimulatory effect of JA on transcriptional activity of the gene encoding proteinase inhibitor. Revealed differences in the activation of defense proteins pointed to differing mechanisms of SA and JA action on protective potential of wheat plants infected with T. caries. [ABSTRACT FROM AUTHOR]

Kazal proteinase inhibitors (KPIs) have been described as important regulators of serine proteinases in invertebrate physiological processes, including the immune response. Several Kazal proteins have been identified in the hemocytes of crustaceans, including freshwater crayfishes, penaeid shrimps, and other shrimps and crabs. Kazal proteins have a repetitive homologous domain structure, and this molecular architecture complicates phylogenetic comparison. Such comparison, with a proper delineation of repeats at the sequence level, however, is important for understanding the structure and function of proteins as well as for the detection of homologous sequences that contribute to explain their evolutionary past. The duplication of coding sequences is a mechanism known to facilitate evolution, and the most important divergence of KPIs appears to be due to insertion, deletion or re-arrangement of complete Kazal domains. A comparison of individual domains, instead of the full protein sequence, thus seems to provide more information and allows a description of the molecular architecture and domain distribution. Considering that each domain undergoes mutation independently, variability in primary sequence via nucleotide substitution is more frequent than the tolerable insertion or deletion of a codon (one amino acid). In addition to primary structure, the distances between Cys residues could therefore be used for domain comparison. Kazal domains from decapod crustaceans are compared using their inter-Cys distances, a nomenclature is proposed for future studies, and the molecular architecture or domain arrangement of these proteins is presented. [ABSTRACT FROM AUTHOR]

Background: Plants 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%. Results: The 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. Conclusion: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta. [ABSTRACT FROM AUTHOR]

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, Dragačevka 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. [ABSTRACT FROM AUTHOR]

Legume proteinase inhibitors (PI/s) abrogate proteolytic activity of proteins and cause adverse effects on growth. In this study, two legume PIs – Archidendron ellipticum Trypsin Inhibitor (AeTI) and Derris trifoliata Trypsin Chymotrypsin Inhibitor (DtTCI) were purified and used along with standard, Soybean Trypsin Inhibitor (STI), either singly or in combination, as antiplasmodial agents against two Plasmodium forms (Pf 3D7/Pf FCR3). Both AeTI/DtTCI were purified to homogeneity by HPLC and showed over 98% trypsin/chymotrypsin inhibition, respectively. DtTCI severely arrested growth of both the Plasmodium forms (IC50 of 9.59 μM and 16.86 μM, respectively). In addition, combination of DtTCI/AeTI had synergistic effect against both Pf 3D7 (FIC – 0.19) and Pf FCR3 (FIC − 0.23). Combinations of DtTCI/STI and AeTI/STI showed additive effects for the parasite forms. Time-course studies indicated DtTCI and combination of DtTCI/AeTI, to be potent inhibitor of schizont rupture. Antiproteolytic action of the PIs on Pf Apical Merozoite Antigen 1 (AMA1) protein revealed that none of the inhibitors (singly/combinations) affected the primary proteolysis of PfAMA1 protein. Notably however, the normal secondary proteolysis of PfAMA1 was abrogated by both DtTCI (singly/combinations) and AeTI. Incidentally, the proteolytic processing of PfAMA1 remained unaffected following STI treatment. [ABSTRACT FROM AUTHOR]

The review summarizes the available published data on proteases and their inhibitors in cestodes. The intricate physiological relationships between a parasite and its host are confirmed by the ability of cestodes to produce their own proteolytic enzymes or to adsorb and utilize host proteases, on the one hand, and to secrete inhibitors of these enzymes, on the other. It is likely that the protease inhibitors primarily confront the host immune response in larval cestodes versus adult worms, living in the vertebrate intestine, where they mainly protect the parasites from hydrolysis by intestinal proteinases. In addition, the presence of the parasite in the intestine often entails changes in the activity of host proteolytic enzymes. [ABSTRACT FROM AUTHOR]

Better knowledge on responses to dehydration stress could help to improve the existing cryopreservation protocols for potato, since plant tissues processed for cryopreservation are often submitted to similar in vitro stress conditions. Cryopreservation (the best method of conservation for vegetatively propagated plants) of potato still needs to be standardized to make it available and to conserve the wide diversity of this crop. In the present work, the response to osmotic stress and chilling temperature was investigated in two potato species, Solanum tuberosum and its relative, frost-tolerant S. commersonii. After 14 days of exposure, different growth parameters, such as shoot length and number of leaves, were measured. Furthermore, differentially abundant proteins were identified after performing 2-fluorescence difference gel electrophoresis (2-DIGE) experiments, and soluble carbohydrates were analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). The results show different responses in both species depending on the stress treatment. Focusing on the differences in growth parameters during the treatments, Solanum commersonii seems to be more affected than S. tuberosum cv. Désirée. At the molecular level, there are some differences and similarities between the two potato species studied that are dependent on the type of stressor.

Responses of resistant (Mi-1/Mi-1) and susceptible (mi-1/ mi-1) tomato (Solanum lycopersicum) to root-knot nematodes (RKNs; Meloidogyne spp.) infection were monitored using cDNA microarrays, and the roles of salicylic acid (SA) and jasmonic acid (JA) defense signaling were evaluated in these interactions. Array analysis was used to compare transcript profiles in incompatible and compatible interactions of tomato roots 24 h after RKN infestation. The jai1 and def1 tomato mutant, altered in JA signaling, and tomato transgenic line NahG, altered in SA signaling, in the presence or absence of the RKN resistance gene Mi-1, were evaluated. The array analysis identified 1,497 and 750 genes differentially regulated in the incompatible and compatible interactions, respectively. Of the differentially regulated genes, 37% were specific to the incompatible interactions. NahG affected neither Mi-1 resistance nor basal defenses to RKNs. However, jai1 reduced tomato susceptibility to RKNs while not affecting Mi-1 resistance. In contrast, the def1 mutant did not affect RKN susceptibility. These results indicate that JA-dependent signaling does not play a role in Mi-1-mediated defense; however, an intact JA signaling pathway is required for tomato susceptibility to RKNs. In addition, low levels of SA might be sufficient for basal and Mi-1 resistance to RKNs.

MMP-9 activity is strongly related to cancer growth and metastization. This study aimed at assessing the inhibitory potential of the major seed protein fractions from eight selected legume species towards MMP-9 activity in colon carcinoma cells. Albumin and globulin fractions were screened for MMP-9 inhibitors, using a fluorometric assay and gelatin zymography. Their effect on HT29 cell proliferation and cell migration was tested, as well as on the corresponding intrinsic cellular MMP-9 activities. Seed proteins include potent inhibitors of MMP-9, particularly low molecular mass proteins. Their effectiveness differs greatly among species, with a positive correlation detected between their inhibitory activity and the reduction in cell migration. Lupin seeds contain the most efficient MMP-9 inhibitors of all legume seeds analyzed, inhibiting both gelatinases and HT29 migration and growth, while pea seeds showed no effect. Results reveal legume protein MMPIs as novel metalloproteinase inhibitors with possible pharmacological interest. This may be important for selecting leguminous species with potential use in anti-cancer diets. [ABSTRACT FROM AUTHOR]

Herbivorous spider mites occurring on tomato plants ( Solanum lycopersicum L.) cope with plant defences in various manners: the invasive Tetranychus evansi reduces defences below constitutive levels, whereas several strains of T. urticae induce such defences and others suppress them. In the Mediterranean region, these two species co-occur on tomato plants with T. ludeni, another closely related spider mite species. Unravelling how this third mite species affects plant defences is thus fundamental to understanding the outcome of herbivore interactions in this system. To test the effect of T. ludeni on tomato plant defences, we measured (1) the activity of proteinase inhibitors, indicating the induction of plant defences, in those plants, and (2) mite performance on plants previously infested with each mite species. We show that the performance of T. evansi and T. ludeni on plants previously infested with T. ludeni or T. evansi was better than on clean plants, indicating that these two mite species down-regulate plant defences. We also show that plants attacked by these mite species had lower activity of proteinase inhibitors than clean plants, whereas herbivory by T. urticae increased the activity of these proteins and resulted in reduced spider mite performance. This study thus shows that the property of down-regulation of plant defences below constitutive levels also occurs in T. ludeni. [ABSTRACT FROM AUTHOR]

Plants respond to attacks by herbivores with various defences, which are mounted through the activation of different biochemical pathways that are known to interact. Thus, the attack of a plant by one herbivore species may result in changes in the performances of other species on the same plant. It has been suggested that species with comparable feeding modes induce similar plant defences and such herbivores are therefore expected to have a negative effect on each other's performance. We studied two closely related phytophagous mite species with identical feeding modes. Yet, one of the species ( Tetranychus urticae) induces tomato plant defences, whereas the other ( T. evansi) reduces them. We found that the 'inducing' species benefits from the downregulation of defences by the 'reducing' species, which, in turn, suffers from the induction of defences by the inducing species. Moreover, the performances of the two mite species on leaves that were previously attacked by both species simultaneously were intermediate between that on leaves previously attacked by each of the mites separately. The activity of proteinase inhibitor, a defensive compound, was not found to be intermediate in leaves attacked by both species simultaneously-it was almost as high as the activity seen in leaves with defences induced by T. urticae. Oviposition rates of T. urticae showed a nonlinear correlation with inhibitor activity, suggesting that it is potentially problematic to use this activity as an indicator of the level of plant defence. Our results show that herbivores with similar feeding modes have opposite effects on plant defence and differentially affect each other's performance on co-infested plants. [ABSTRACT FROM AUTHOR]

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Proteinase activity in the midgut of the pentatomid stinkbug Podisus maculiventris was investigated. The optimal pH for adult and nymph proteolysis was pH 6.0 and pH 6.5, respectively. Proteinase activity was characterised using a range of diagnostic inhibitors. Activity of both adult and nymphal gut extracts, detected by the hydrolysis of Z-Phe-Arg-pNA, was inhibited to

Rationale: The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. Objectives: To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. Methods: Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. Main Results: Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-877 to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01). Conclusions: Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-877 by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia. [ABSTRACT FROM AUTHOR]

Plants are continuously challenged by several environmental stresses that impair their growth and production performances. Stresses encountered by plants can be caused by biotic agents (e.g., herbivores, parasitic microorganisms, weeds) and abiotic factors (e.g., cold, drought, soil and water salinity). Despite these differences, plants respond to biotic and abiotic stresses with shared adaptive mechanisms resulting in complex and interlinked cross-talks. Systemin is a hormone peptide, playing a central role in the regulation of plant response against a wide range of stress agents, including wound, phytopathogenic fungi and phytophagous and sucking insects. It has also been shown that upregulation of prosystemin, the precursor protein of systemin, enhanced the tolerance of tomato plants to salt stress, indicating that systemin induced molecular adaptations to biotic stress can also be beneficial to plants exposed to salt stress. Considering that systemin is a small peptide that can be sensed by plants both as soil drench and foliar applications, we hypothesized that exogenous applications of systemin may increase salt stress tolerance in tomato plants through the activation of multiple adaptation mechanisms. Here we report that a soil drench picomolar solution of systemin increases tomato salt stress tolerance by i) activating of SOS1 , NHX and HKT Na+ transporters in leaves ii) enhancing the cellular antioxidant power, and iii) balancing the protease activity induced by salt stress. Activation of these responses upon exogeneous application of systemin was highly correlated to an improved tomato growth under salt stress, suggesting that systemin may represent an important component of the crosstalk between biotic and abiotic stress responses in plants. • Exogenous treatment with systemin enhances salt stress tolerance in tomato plants. • Systemin improved osmotic balance, antioxidant activity, and protein stability. • Systemin mediates common adaptation mechanisms to biotic/abiotic stresses. • Biotic/abiotic stress crosstalk could be the target for future plant protection. [ABSTRACT FROM AUTHOR]

One of the most important roles of proteins in cellular milieu is recognition of other biomolecules including other proteins. Protein–protein complexes are involved in many essential cellular processes. Interfaces of protein–protein complexes are traditionally known to be conserved in evolution and less flexible than other solvent interacting tertiary structural surface. But many examples are emerging where these features do not hold good. An understanding of inter-play between flexibility and sequence conservation is emerging, providing a fresh dimension to the paradigm of sequence–structure–function relationship. The functional manifestation of the inter-relation between sequence conservation and flexibility of interface is exemplified in this review using proteinase–inhibitor protein complexes. [ABSTRACT FROM AUTHOR]

The molecular interactions between pancreatic lipase (PL) and four tea polyphenols (EGCG analogs), like (−)-epigallocatechin gallate (EGCG), (−)-gallocatechin gallate (GCG), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin (EC), were studied from PL activity, conformation, kinetics and thermodynamics. It was observed that EGCG analogs inhibited PL activity, and their inhibitory rates decreased by the order of EGCG>GCG>ECG>EC. PL activity at first decreased rapidly and then slowly with the increase of EGCG analogs concentrations. α-Helix content of PL secondary structure decreased dependent on EGCG analogs concentration by the order of EGCG>GCG>ECG>EC. EGCG, ECG, and EC could quench PL fluorescence both dynamically and statically, while GCG only quenched statically. EGCG analogs would induce PL self-assembly into complexes and the hydrodynamic radii of the complexes possessed a close relationship with the inhibitory rates. Kinetics analysis showed that EGCG analogs non-competitively inhibited PL activity and did not bind to PL catalytic site. DSC measurement revealed that EGCG analogs decreased the transition midpoint temperature of PL enzyme, suggesting that these compounds reduced PL enzyme thermostability. In vitro renaturation through urea solution indicated that interactions between PL and EGCG analogs were weak and non-covalent. [ABSTRACT FROM AUTHOR]

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin ( Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain ( Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target. Findings: Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range. Conclusions: Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications. [ABSTRACT FROM AUTHOR]

Targeting allosteric sites is gaining increasing recognition as a strategy for modulating the activity of enzymes, especially in drug design. Here we investigate the mechanisms of allosteric regulation of cathepsin K as a representative of cysteine cathepsins and a promising drug target for the treatment of osteoporosis. Eight novel modifiers are identified by computational targeting of predicted allosteric sites on the surface of the enzyme. All act via hyperbolic kinetic mechanisms in presence of low molecular mass substrates, as expected for allosteric effectors. Two compounds have sizable effects on enzyme activity using interstitial collagen as a natural substrate of cathepsin K and four compounds show a significantly stabilizing effect on cathepsin K. The concept of activity modification space is introduced to obtain a global perspective of the effects elicited by the modifiers. Analysis of the activity modification space reveals that the activity of cathepsin K is regulated via multiple, different allosteric mechanisms. [ABSTRACT FROM AUTHOR]

Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10−5 M−1s−1, for rSW-AT-1 and chymotrpsin is 2.85×10−6 M−1s−1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4–10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed. [ABSTRACT FROM AUTHOR]

Highlights: [•] Inhibitor sequence variations lead to variance in inhibition and specificity. [•] Differential inhibition offered alteration in digestive physiology. [•] Performance variability can be explored for designing new effective inhibitor. [ABSTRACT FROM AUTHOR]

ABSTRACT This article describes the synthesis and enzymatic study of newly synthesized analogs of trypsin inhibitors SFTI-1 that were fluorescent labeled on their N-terminal amino groups. Two fluorescent derivatives of benzoxazole (3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]- l-alanine-[(4NPh2)Ph]Box-Ala and 3-[ 2-( 2', 4',5'-t rimethoxyphenyl) benzoxazol-5-yl]-l- alanine-[2,4,5-(OMe)3Ph]Box-Ala) were used as efficient fluorescent labels. The compounds obtained preserved their inhibitory activity and were efficient inhibitors of bovine trypsin or chymotrypsin. Nevertheless, their association inhibition constants were one or two orders of magnitude lower than those determined for unlabeled monocyclic SFTI-1 or [Phe5]SFTI-1, respectively. The conjugates obtained were found to be proteolytically stable in the presence of cognate enzymes. Applying such fluorescent peptides, we were able to investigate enzyme-inhibitor complex formation using fluorescent techniques. We found that such compounds were rapidly internalized by the fibroblast or cancer cells with no cytotoxic effects. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 124-135, 2014. [ABSTRACT FROM AUTHOR]

This paper presents the results of a structure-functional study of neurotoxins, actinoporins, and proteinase inhibitors from the tropical sea anemone Heteractis crispa (= Radianthus macrodactylus) and the low boreal sea anemone Oulactis orientalis. The new data significantly contribute to the knowledge of the mechanisms of action of polypeptides from marine coelenterates and can be useful for solving practical problems of the biotechnological development of medicines. [ABSTRACT FROM AUTHOR]

Helicoverpa armigerais a polyphagous pest damaging vast numbers of different crops leading to decrease in total production. Use ofBttransgenic to controlH. armigerahas worked well but has increased resistance againstBtinH. armigeraand controversies about theBttransgenic making it imperative to find another strategy to control attack. Soybean is a nonhost plant forH. armigera; reason could be laid in the defense system of the soybean. Proteinase Inhibitor (PIs) have been extensively studied for development of resistance against insect pest. Two cultivars developed by our university were investigated for the presence of proteinase inhibitors namely, MAUS-158 and MAUS-61. Partially purified inhibitors were showed inhibition of total protease activity of gut extract by 91.34±1.49 and 89.95±0.96% by MAUS-158 and MAUS-61, respectively. While inhibition of trypsin like proteases were found between 65 and 71% and inhibition of chymotrypsin like proteases ranges between 40 and 42%. The partial purification study shows stability of PIs up to 60°C. Soybean PIs are also showing more prominent inhibition pattern against trypsin than chymotrypsin. [ABSTRACT FROM AUTHOR]

Plant proteinase inhibitors (PIs) have been shown to reduce the growth rates in larvae of numerous insect species. On the other hand, insects can also regulate their proteinases against plant PIs. In the present study, we report the compensatory activities of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) gut proteinases against the PIs of Albizia lebbeck seeds. Total of ten proteinase inhibitor bands were detected in the seed extract of A. lebbeck. Bioassays were conducted by feeding H. armigera larvae on diet containing partially purified PIs from A. lebbeck seeds. Results show that larval growth and survival was significantly reduced by A. lebbeck PIs. We found that higher activity H. armigera gut proteinase (HGP) isoforms observed in the midgut of control larvae were inhibited in the midgut of larvae fed on test diet. Some HGP isoforms were induced in the larvae fed on PI containing test diet; however, these isoforms showed lower activity in the larvae fed on control diet. Aminopeptidase activities were significantly increased in the midgut of larvae fed on test diet. A population of susceptible and resistant enzymes was observed in the midgut of H. armigera, when fed on diet containing PIs from A. lebbeck seeds. Our initial observations indicate that H. armigera can regulate its digestive proteinase activity against non-host plant PIs, too. It is important to study the exact biochemical and molecular mechanisms underlying this phenomenon in order to develop PI-based insect control strategies. [ABSTRACT FROM AUTHOR]

Better knowledge on responses to dehydration stress could help to improve the existing cryopreservation protocols for potato, since plant tissues processed for cryopreservation are often submitted to similar in vitro stress conditions. Cryopreservation (the best method of conservation for vegetatively propagated plants) of potato still needs to be standardized to make it available and to conserve the wide diversity of this crop. In the present work, the response to osmotic stress and chilling temperature was investigated in two potato species, Solanum tuberosum and its relative, frost-tolerant S. commersonii. After 14 days of exposure, different growth parameters, such as shoot length and number of leaves, were measured. Furthermore, differentially abundant proteins were identified after performing 2-fluorescence difference gel electrophoresis (2-DIGE) experiments, and soluble carbohydrates were analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). The results show different responses in both species depending on the stress treatment. Focusing on the differences in growth parameters during the treatments, Solanum commersonii seems to be more affected than S. tuberosum cv. Désirée. At the molecular level, there are some differences and similarities between the two potato species studied that are dependent on the type of stressor. [ABSTRACT FROM AUTHOR]