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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]

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]

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]

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]

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]

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]

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]

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]

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]

Summary: We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection. [ABSTRACT FROM AUTHOR]

Protease inhibitors (PIs) retard growth and development and cause mortality in a range of insect pests belonging to various orders. Some transgenic plants expressing PI genes have also been effective against insect pest attack. The effects of two purified soybean PIs, i.e. trypsin inhibitor (Kunitz type; SBTI) and trypsin–chymotrypsin inhibitor (Bowman-Birk type; SBBI), were investigated on first and third instar larvae of the melon fruit fly Bactrocera cucurbitae (Coquillett) in laboratory feeding bioassays. The larval period of both the first and third instar larvae was prolonged when the larvae were treated with various concentrations of SBTI and SBBI. The total development period prolonged significantly in both the instars of larvae treated with SBTI, but in SBBI-treated larvae, the development period was delayed significantly only in the third instar larvae. A decrease in percentage pupation and adult emergence was observed in the first and third instar larvae treated with different concentrations of both SBTI and SBBI. The present studies showed that SBTI and SBBI have a potential in inhibiting the growth and development of melon fruit fly, but further detailed and molecular level studies are required before their use in transgenics. [ABSTRACT FROM AUTHOR]

Previous work identified aphids and caterpillars as having distinct effects on plant responses to herbivory. We sought to decipher these interactions across different levels of biological organization, i.e., molecular, biochemical, and organismal, with tomato plants either damaged by one 3rd-instar beet armyworm caterpillar ( Spodoptera exigua), damaged by 40 adult potato aphids ( Macrosiphum euphorbiae), simultaneous damaged by both herbivores, or left undamaged (controls). After placing insects on plants, plants were transferred to a growth chamber for 5 d to induce a systemic response. Subsequently, individual leaflets from non-damaged parts of plants were excised and used for gene expression analysis (microarrays and quantitative real-time PCR), C/N analysis, total protein analysis, proteinase inhibitor (PI) analysis, and for performance assays. At the molecular level, caterpillars up-regulated 56 and down-regulated 29 genes systemically, while aphids up-regulated 93 and down-regulated 146 genes, compared to controls. Although aphids induced more genes than caterpillars, the magnitude of caterpillar-induced gene accumulation, particularly for those associated with plant defenses, was often greater. In dual-damaged plants, aphids suppressed 27% of the genes regulated by caterpillars, while caterpillars suppressed 66% of the genes regulated by aphids. At the biochemical level, caterpillars induced three-fold higher PI activity compared to controls, while aphids had no effects on PIs either alone or when paired with caterpillars. Aphid feeding alone reduced the foliar C/N ratio, but not when caterpillars also fed on the plants. Aphid and caterpillar feeding alone had no effect on the amount of protein in systemic leaves; however, both herbivores feeding on the plant reduced the amount of protein compared to aphid-damaged plants. At the organismal level, S. exigua neonate performance was negatively affected by prior caterpillar feeding, regardless of whether aphids were present or absent. This study highlights areas of concordance and disjunction between molecular, biochemical, and organismal measures of induced plant resistance when plants are attacked by multiple herbivores. In general, our data produced consistent results when considering each herbivore separately but not when considering them together. [ABSTRACT FROM AUTHOR]

Abstract: Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel’s foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K i, 0.1×10−9 M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. [Copyright &y& Elsevier]

Depending on the stress type, plants activate various signal transduction pathways inducing the optimum defense process. This review is devoted to jasmonate (JA) dependent signaling involved in plant defense against biotic and abiotic stresses, including those determined by wounding, necrotrophic pathogens, pests, and herbivores. The sequence of major events of JA signaling is discussed. It is noted that JA signaling in plants is incorporated into a complex signaling network. [ABSTRACT FROM AUTHOR]

Screening of genetically enhanced cotton varieties with a high content of protein markers, proteinase inhibitors, was carried out for the first time. It was found that only 5 of 68 studied cotton selection lines were resistant to wilt, black root rot, and gummosis. [ABSTRACT FROM AUTHOR]

Abstract: Representative species, two from each of the cockroach families Blattidae, Blattellidae, and Blaberidae, have similar morphology of the digestive tract but differ in the physiology of digestion. The pH of crop and along the midgut varies in different species from 5.9 to 9.0 and the redox parameter from 10.1 to 12.9. Activities of proteinases and amylases in comparable gut regions differ among the species up to 100 times. Proteolytic activity is high in the midgut and moderate in the crop of Blattidae; in the other species, it is very low in the crop and increases to a moderate level in the posterior half of midgut (PM). The level of amylolytic activity is similar in the examined gut compartments of Blattidae and Blattellidae but low in the PM of Blaberidae. Blaberidae are also characterized by a high potential of the salivary glands, crop, and midgut to inhibit subtilisin, trypsin, and chymotrypsin. Inhibition of these proteinases by the extracts of salivary glands and gut is several orders of magnitude lower and often undetectable in the representatives of Blattidae and Blattellidae. [Copyright &y& Elsevier]

The article reports on the rice plants' pseudomonas-induced defence molecules against leaffolder (Cnaphalocrocis medinalis) pest. In relation, evaluation of the fluorescent pseudomonad strains Pf1, PY15 and TDK1 individually and in combination were conducted. An overview of the various treatments used is offered. It also demonstrates the induction of defense-related molecules. High accumulation of defense molecules such as proteinase and chitinase inhibitors were observed with a combined TDK1, Pf1 and PY15 treatment compared with all other treatments. As a result of the study, a combination of fluorescent pseudomonad strains affects the development of leaffolder pest by inducing defense molecules in rice plants which in turn enhance resistance to leaffolder attack is revealed.

Abstract: Although our understanding of the molecular pathogenesis of common types of cancer has improved considerably, the development of effective strategies for cancer diagnosis and treatment have lagged behind. Mouse models of cancer potentially represent an efficient means for uncovering diagnostic markers as genetic alterations associated with human tumors can be engineered in mice. In addition, defined stages of tumor development, breeding conditions, and blood sampling can all be controlled and standardized to limit heterogeneity. Alternatively human cancer cells can be injected into mice and tumor development monitored in xenotransplants. Mouse-based studies promise to elucidate a repertoire of protein changes that occur in blood and biological fluids during tumor development. This is illustrated in a study in which we have applied a three-dimensional intact protein analysis system (IPAS) to elucidate detectable protein changes in serum from immunodeficient mice with lung xenografts from orthotopically implanted human A549 lung adenocarcinoma cells. With sufficiently detailed protein sequence identifications, the observed protein changes can be attributed to either the host mouse or the human tumor cells. It is noteworthy that the majority of increases identified have corresponded to relatively abundant serum proteins, some of which have previously been reported as increased in the sera of cancer patients. Proteomic studies of mouse models of cancer allow assessment of the range of changes in plasma proteins that occur with tumor development and may lead to the identification of potential cancer markers applicable to humans. [Copyright &y& Elsevier]

Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma. These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage. Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously trafficked to two separate compartments mediated by proteolysis early in the secretory pathway. [ABSTRACT FROM AUTHOR]

Abstract: Background: The matrix metalloproteinase (MMP) system is responsible for degradation of tissue in both normal and pathological processes, including tumour invasion and metastasis. Aim: To compare tissue concentrations of components of the MMP system between tumour tissue and normal tissue in patients with oral squamous cell carcinoma, and to correlate concentrations with pathological grade of tumour. Methods: Thirty-eight paired tissue samples from tumours and normal tissue were analysed by three laboratory techniques: firstly, enzyme linked immunosorbent assays (ELISA) in ng/mg protein for MMP-1, MMP-3, and tissue inhibitors of metalloproteinases (TIMPs) -1 and -2. Secondly, gelatinase activity assays to measure concentrations of total and endogenous active gelatinases, MMP-2 and MMP-9 (ng/mg protein). And thirdly to use quenched fluorescent substrate hydrolysis to measure total MMP activity (pM/min). Results: The concentration of all MMPs was significantly higher in tumour than in normal oral tissue (p <0.05, Mann–Whitney U-test). Tissue concentrations of some of these factors correlated with clinical and pathological indices of aggressiveness of tumours, including T-stage, N-stage, tumour differentiation, and anatomical level of involved nodes. However, the study was not powered to show statistical significance. Conclusion: It is the balance between proteinases and their inhibitors that controls tissue degradation at each stage of tumour invasion and metastasis. Measurement of MMPs in oral mucosal biopsy samples may establish the invasive potential of tumours at their initial presentation. [Copyright &y& Elsevier]

Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a ‘mouse trap’ against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato ( Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media. [ABSTRACT FROM AUTHOR]

Plants release volatile organic compounds (VOCs) in response to wounding and herbivore attack, some of which trigger responses in neighboring unattacked plants in the laboratory under conditions that are not likely to occur in the real world. Whether plants ‘eavesdrop’ on the volatile emissions of their neighbors in nature is not known. The best documented field study of between-species signaling via above-ground VOCs involves increases in fitness parameters of native tobacco ( Nicotiana attenuata) transplanted adjacent to clipped sagebrush ( Artemesia tridentata tridentata). Clipped sagebrush releases many biologically active VOCs, including methyl jasmonate (MeJA), methacrolein and a series of terpenoid and green leaf VOCs, of which MeJA, while active under laboratory conditions, is not released in sufficient quantities to directly elicit induced resistance in the field. Here we demonstrate, with laboratory and field-based experiments, that priming (rather than direct elicitation) of native N. attenuata’s induced chemical defenses by a sagebrush-released VOC bouquet can account for earlier findings. With microarrays enriched in N. attenuata herbivore-regulated genes, we found transcriptional responses in tobacco growing adjacent to clipped sagebrush foliage, but failed to detect the direct elicitation of defensive chemicals or proteins. However, we observed an accelerated production of trypsin proteinase inhibitors when Manduca sexta caterpillars fed on plants previously exposed to clipped sagebrush. This readying of a defense response, termed priming, results in lower total herbivore damage to plants exposed to clipped sagebrush and in a higher mortality rate of young Manduca caterpillars. Our study demonstrates priming of plant defense responses as a mechanism of plant–plant signaling in nature, and provides an example for the analysis of between-plant signaling under ecologically realistic conditions. Although we describe priming as a potential mechanism for signaling between plants in nature, we critically discuss the ecological relevance of the particular interaction. [ABSTRACT FROM AUTHOR]

Proteinases and their inhibitors play important roles in neural development, homeostasis and disease. Neuroserpin is a member of the serine proteinase inhibitor (serpin) superfamily that is secreted from the growth cones of neurons and inhibits the enzyme tissue-type plasminogen activator (tPA). The temporal and spatial pattern of neuroserpin expression suggests a role in synaptogenesis and is most prominent in areas of the brain that participate in learning, memory and behaviour. Neuroserpin also provides neuronal protection in pathologies such as cerebral ischaemia and epilepsy by preventing excessive activity of tPA. Point mutations in neuroserpin cause aberrant conformational transitions and the formation of loop-sheet polymers that are retained within the endoplasmic reticulum of neurons, forming inclusion bodies that underlie an autosomal dominant dementia that we have called familial encephalopathy with neuroserpin inclusion bodies or FENIB. We review here the role of neuroserpin and other proteinase inhibitors in brain development, function and disease. [ABSTRACT FROM AUTHOR]

In this work we evaluate the proteolytic state of plasma α2-macroglobulin in acute pancreatitis. In addition, the plasma activity of matrix metalloproteinase-2 (MMP-2), MMP-9 and serine proteinases were analyzed. A total of 33 patients with acute pancreatitis were studied, of whom 16 were diagnosed as having mild and 17 as having severe acute pancreatitis. In the latter group, three patients progressed to multi-organ failure and died as a consequence of these complications. The proteolytic fragmentation of α2-macroglobulin was evaluated by Western blotting, whereas the plasma activity of MMP-2, MMP-9 and serine proteinases was evaluated by gelatin zymography. Enhanced fragmentation of α2-macroglobulin was detected in severe acute pancreatitis patients with multiple organ failure and lethal complications. In this same patient group, increased plasma activity of the active forms of MMP-2 and MMP-9, as well as serine proteinases, was apparent. In addition, we demonstrate that chymotrypsin-like proteinases could be the principal cause of α2-macroglobulin degradation in this group of patients. Our results indicate that secondary proteolysis of α2-macroglobulin promotes impaired control of extracellular proteolytic activity, leading to local and distant tissue injuries during severe acute pancreatitis. Finally, the structural evaluation of plasma α2-macroglobulin could be used as a prognostic marker of the severity of acute pancreatitis. [ABSTRACT FROM AUTHOR]

The effect of ritonavir and saquinavir, HIV proteinase inhibitors, on the secreted aspartyl proteinase (Sap) activity of Candida parapsilosis was studied. In a proteinase-inducing medium (yeast carbon base-bovine serum albumin), Sap activity in all clinical isolates of C. parapsilosis (n = 20) was observed at 37°C but not at 22°C. The presence of ritonavir at a concentration of 8 μg/ml produced an inhibition close to 50% albumin consumption and also delayed yeast growth; however, saquinavir did not have any effect on growth or on Sap activity. In Sabouraud broth, which does not induce Sap production, no effect was shown on yeast growth by either of the two HIV proteinase inhibitors studied. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation. [ABSTRACT FROM AUTHOR]

Herbivore-induced plants responses can affect the preference and performance of herbivores and their natural enemies. These responses may vary depending on the identity and number of herbivore species feeding on the plant so that when herbivores from different guilds feed on plants, the interactions between plants, herbivores, and natural enemies may be disrupted. Tomato plants were damaged either by the caterpillar Spodoptera exigua, or the aphid Macrosiphum euphorbiae, or damaged by both herbivores, or undamaged controls. We measured the preference and performance of S. exigua and its parasitoid Cotesia marginiventris, and activity of proteinase inhibitors (PI) as an indicator of induced resistance. Compared to undamaged plants, caterpillar damage reduced the number of eggs laid by S. exigua adults, reduced growth, consumption, and survival of larval S. exigua and C. marginiventris, and increased activity of PIs 43%; but did not increase attraction of C. marginiventris. While pupal mass of S. exigua was not affected, the pupal mass of C. marginiventris decreased on caterpillar-damaged plants compared to controls. In contrast, plants damaged by aphids were preferred for oviposition by S. exigua, and had increased larval consumption and survival, compared to controls. Aphid feeding did not affect the preference or performance of C. marginiventris, or PI activity, compared to controls. While oviposition was deterred on caterpillar-damaged plants, plants damaged by both herbivores received the same amount of oviposition as controls. The attraction of C. marginiventris to plants damaged by caterpillars and aphids was increased compared to controls. However, plants damaged by both herbivores had similar PI activity, larval growth and survival of S. exigua and C. marginiventris, as plants singly damaged by caterpillars. Overall, the preference component for both the herbivore and parasitoid was more strongly affected by damage due to multiple herbivores than the performance component. [ABSTRACT FROM AUTHOR]

The study evaluated the quality of plasma obtained after whole-blood filtration with four different polyester filters and one polyurethane filter. The activities of coagulation factors and proteinase inhibitors were not or only negligibly affected by filtration, in all experiments. Filtration did not increase markers of clotting and fibrinolysis. Only a strong neutrophil and complement activation was observed, which depended on the type of filter and whole-blood storage conditions. However, as neutrophil elastase-specific degradation products did not increase and the complement-derived anaphylatoxin C3a was found in its inactivated form, C3a-desArg, these filtration-dependent changes apparently have little impact on the therapeutic quality of whole-blood-filtered fresh frozen plasma for transfusion. [ABSTRACT FROM AUTHOR]

Highly active anti-retroviral therapies (HAART) with human immunodeficiency virus (HIV) protease inhibitors (PIs) or nonnucleoside reverse-transcriptase inhibitors (NNRTI) were compared for their effect on prevalence, aspartyl proteinase (Sap) production and the biotypes and anti-mycotic sequential susceptibility of Candida spp. isolates from the oral cavity in a longitudinal prospective study. HAART-PI, but not HAART-NNRTI strongly inhibited Sap expression in the oral cavity without exerting any consistent effect on the role of Candida spp. isolation or selection of low virulence or anti-mycotic resistant fungus biotype. More importantly, the sequential isolates of Candida albicans from HAART-PI, but not those from suspended HAART-NNRTI, showed an increased Sap production in vitro. While further demonstrating that HIV-PI inhibit Sap expressions, our results do not support the view that the mentioned inhibition could eliminate Candida or its selection of the oral cavity. [Copyright &y& Elsevier]

Genetically modified (GM) potatoes expressing a cysteine proteinase inhibitor (cystatin) have been developed as an option for the management of plant parasitic nematodes. The relative impact of such plants on predators and parasitoids (natural enemies) of nontarget insects was determined in a field trial. The trial consisted of GM plants, control plants grown in soil treated with a nematicide and untreated control plants. The quantity of nontarget aphids and their quality as hosts for natural enemies were studied. Aphid density was significantly reduced by nematicide treatment and few natural enemies were recorded from treated potatoes during the study. In contrast, similar numbers of aphids and their more adundant predators were recorded from the untreated control and the GM potatoes. The size of aphids on GM and control plants was recorded twice during the study. During the first sampling period (2–9 July) aphids clip-caged on GM plants were smaller than those on control plants. During the second sampling period (23–30 July) there was no difference in aphid size between those from the GM and control plants. Host size is an important component of host quality. It can affect the size and fecundity of parasitoid females and the sex ratio of their offspring. However, neither the fitness of females of Aphidius ervi, the most prevalent primary parasitoid, nor the sex ratio of their progeny, were affected when the parasitoids developed on aphids feeding on GM plants. Two guilds of secondary parasitoid were also recorded during the study. The fitness of the most abundant species, Aspahes vulgaris, was not affected when it developed on hosts from GM plants. The transgene product, OC IΔD86, was not detected in aphids that had fed on GM plants in the field, suggesting that there is minimal secondary exposure of natural enemies to the inhibitor. The results indicate that transgenic nematode resistance is potentially more compatible with aphid biological control than is current nematicide use. [ABSTRACT FROM AUTHOR]

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (Mr>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with Mr 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown. [Copyright &y& Elsevier]

Background: Antimicrobial agents are sometimes used as adjuncts for the treatment of aggressive and refractory forms of periodontitis. In this study, we used a culture plate assay to investigate the effect of suboptimal doses of antimicrobial agents on proteinase activity of Porphyromonas gingivalis.Methods: A culture plate assay using gelatin as the substrate, which allows a semiquantitative determination of proteinase activity, was developed. Suboptimal inhibitory concentrations of tetracycline, minocycline, doxycycline, metronidazole, penicillin G, or chlorhexidine were added to the medium, and proteolysis zones were determined following the growth of three strains of P. gingivalis. The effect of antimicrobials on outer membrane vesicle-associated gingipains also was determined.Results: The gelatin plate assay was a convenient, simple procedure for investigating the effect of suboptimal inhibitory concentrations of antimicrobial agents on proteinases produced by P. gingivalis. The largest reduction (> 75%) in the proteolysis zones produced by three strains of P. gingivalis was obtained with minocycline. Tetracycline and doxycycline also reduced the proteolysis zones. A suboptimal inhibitory concentration of chlorhexidine increased the proteolysis zones by up to 70%. Metronidazole and penicillin G produced no noticeable effect. The suboptimal inhibitory concentrations of minocycline, tetracycline, and doxycyline did not reduce the activity of outer membrane vesicle-associated Arg- and Lys-gingipains.Conclusion: Results from this study suggest that sublethal concentrations of some antimicrobial agents in subgingival sites have the potential to affect the physiology of P. gingivalis, notably by increasing or decreasing the proteolytic activity of the bacteria. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants. [Copyright &y& Elsevier]

A series of myoepithelial cell lines and xenografts derived from benign human myoepithelial tumors of diverse sources (salivary gland, breast, and lung) exhibit common mRNA expression profiles indicative of a tumor-suppressor phenotype. Previously established myoepithelial cell lines and xenografts (HMS-#; HMS-#X) were compared to nonmyoepithelial breast carcinoma cells (MDA-MB-231 and MDA-MB-468, and inflammatory breast carcinoma samples, IBCr, and IBCw), a normal mammary epithelial cell line (HMEC) and individual cases of human breast cancer (zcBT#T), and matched normal human breast tissues (zcBT#N) (overall samples = 22). The global gene expression profile (22,000 genes) of these individual samples was examined using Affymetrix Microarray Gene Chips and subsequently analyzed with both Affymetrix and DChip algorithms. The myoepithelial cell lines/xenografts were distinct and very different from the nonmyoepithelial breast carcinoma cells and the normal breast and breast tumor biopsies. Two hundred and seven specifically selected genes represented a subset of genes that distinguished (P < 0.05) all the myoepithelial cell lines/xenografts from all the other samples and which themselves exhibited hierarchical clustering. Further analysis of these genes revealed increased expression in genes belonging to the classes of extracellular matrix proteins, angiogenic inhibitors, and proteinase inhibitors and decreased expression belonging to the classes of angiogenic factors and proteinases. Developmental genes were also differentially expressed (either over or underexpressed). These studies confirm our previous impression that human myoepithelial cells express a distinct tumor-suppressor phenotype. [Copyright &y& Elsevier]

In response to insect attack, high levels of proteinase inhibitors are synthesised in potato leaves. This can cause inefficient protein digestion in insects, leading to reduced growth, delayed development and lower fecundity. It has been suggested that Colorado potato beetle overcomes this defence mechanism by inducing the production of a set of cysteine proteases that are resistant to potato proteinase inhibitors. Experiments with gut extracts showed that these proteases have unusual inhibition profiles as they are not inhibited by most of the cystatins but are strongly inhibited by thyropins. In this study we have isolated three cysteine proteases from adapted guts of Colorado potato beetle larvae, named intestains 1, 2 and 3, the first cysteine proteases known to be involved in extracellular protein digestion. The N-terminal sequences suggest their classification into the papain family. Intestains differ in substrate specificities and inhibitory profiles. Their substrate specificities suggest that intestains 1 and 2 are general digestive enzymes, while intestain 3 has a more specific function. The inhibitory profile of intestain 1 is similar to that of proteases of the papain family. However, the K[subi] values for the interaction of intestain 2 with the same set of inhibitors are several hundred fold higher, which would enable the enzyme to circumvent the potato defence mechanism characterised by high concentrations of protease inhibitors in attacked potato leaves. A further, different strategy of the Colorado potato beetle to avoid potato defence is exhibited by intestain 3, which is able to cleave off the N-terminus of model cystatin and thus inactivate the inhibitor. These results suggest that the Colorado potato beetle combines different strategies to counteract plant defence mechanisms. [ABSTRACT FROM AUTHOR]

Studies of induced plant resistance usually either examine physiological/chemical mechanisms or explore the ecological and evolutionary role of induced resistance. To connect these two methods of study, data are needed that address the relationships between plant chemistry and effects of induced resistance on herbivores under field conditions. In this paper we combine the results of a greenhouse experiment and a field experiment to try to make such a connection. Levels of induced and constitutive resistance to Mexican bean beetles in several soybean genotypes were measured in a greenhouse experiment using a behavioral bioassay. In a field experiment, beetle performance and induced and constitutive levels of cystein proteinase inhibitor activity were measured for these same genotypes. Greenhouse bioassay ratings of induced and constitutive resistance and induced and constitutive levels of proteinase inhibitor activity agree closely for individual genotypes, suggesting that proteinase inhibitors (PIs) are involved in induced resistance. However, while greenhouse bioassay measures of constitutive and induced resistance were good predictors of beetle performance in the field, proteinase inhibitor activity did not predict beetle performance well. While PIs appear to be involved in induced resistance, they were not strongly correlated to Mexican bean beetle performance in the field in this study. These results suggest that measurement of single aspects of plant chemistry may not encompass all ecologically important aspects of plant resistance. [ABSTRACT FROM AUTHOR]

Background: Tetracyclines, particularly doxycycline (Doxy), and their non-antimicrobial chemically-modified derivatives (CMTs) inhibit the activities of human matrix metalloproteinases (MMPs), and reduce the severity and progression of periodontal disease in animal models and humans. In this study, the effects of Doxy and CMT-1, -3, and -5 on proteolytic, serpinolytic, and progelatinase-B activation activities of potent periodontopathogens were studied.Methods: The effect of Doxy and CMTs (0.5 to 50 microM) on proteolytic activities were investigated by incubating bacteria with chromogenic substrates or human serum albumin. A collagenolytic fraction of Porphyromonas gingivalis was used to evaluate the effect of these substances on collagenolytic (type I collagen) and serpinolytic (alpha1-proteinase inhibitor) activities. Lastly, the effect of Doxy on progelatinase-B (pro-MMP-9) activation by purified proteinases from P. gingivalis and Treponema denticola was investigated by SDS-PAGE/Western immunoblotting.Results: Doxy and CMTs, except CMT-5 which lacks the structural elements required for cation chelation, inhibited Arg- and Lys-gingipain activities as well as collagenolytic activity of P. gingivalis. Doxy and CMTs did not markedly affect the chymotrypsin-like activity of T. denticola but inhibited its trypsin-like activity. In addition, degradation of human serum albumin by cells of P. gingivalis and T. denticola was strongly inhibited by Doxy and CMT-1. Doxy and CMT-1 also inhibited the inactivation of alpha1-proteinase inhibitor (serpinolytic activity) by a collagenolytic fraction of P. gingivalis. Lastly, Doxy prevented the latent to active conversion of human neutrophil progelatinase-B (pro-MMP-9) by Arg-gingipains A/B of P. gingivalis but not by the chymotrypsin-like proteinase of T. denticola.Conclusions: Data from this study suggest that Doxy and CMTs have the potential to inhibit the periodontopathogenic bacterial proteinases, which contribute to tissue destruction cascades during periodontitis directly and indirectly by triggering the host response. [ABSTRACT FROM AUTHOR]

Background: Dipeptide bestatin has been previously reported to selectively inhibit the growth of Porphyromonas gingivalis. The aims of this study were to investigate the mechanism of action of bestatin and to evaluate its effect on epithelial cells. Methods: The inhibitory effect of bestatin on P. gingivalis was tested in vitro (culture medium) and in vivo (guinea pig model). Radiolabeled compounds were used to investigate the effect of bestatin on the uptake of amino acids and peptides. The cytotoxic effect of bestatin was evaluated using a keratinocyte cell Results: The growth inhibition of P. gingivalis by bestatin was concentration-de pendent. Even at high concentrations, compounds possessing a chemical structure or an aminopeptidase inhibitor activity related to bestatin had no effect on growth of P. gingivalis. When injected in the presence of P. gingivalis, bestatin was able to prevent the development of a necrotic abscess in a guinea pig model. Data were obtained suggesting that bestatin does not act on proteinases of P. gingivalis. Rather, bestatin was found to inhibit the intracellular uptake of radioactivity from 14C-labeled amino acids or heat-denatured type I collagen. This was not observed with a spontaneous mutant of P. gingivalis, whose growth was not affected by bestatin. In the second part of the study, bestatin was found to have no effect on epithelial cell viability in culture at concentrations effective on P. gingivalis. In addition, bestatin did not show effects on epithelial cell migration or production of gelatinases. Conclusions: This study suggests that bestatin selectively inhibits growth of P. gingivalis by affecting the intracellular uptake of amino acids and peptides, which serve as energy and nitrogen sources for this bacterial species. Bestatin has no cytotoxicity and may represent a therapeutic molecule for local treatment of P gingivalis-associated periodontitis. [ABSTRACT FROM AUTHOR]

The inhibitory activities of the new gossypol-free cotton variety Bukhara-9 and varieties UzFA-705 and Mirishkor were compared. It was found that Bukhara-9 and UzFA-705 had the same inhibitory activity as the resistant regionalized variety An-Bayaut-2. The inhibitory activity of Mirishkor was greater than that of variety Turon, which was developed for growing in arid zones. It was shown that Mirishkor proteinase inhibitors suppressed practically fully growth in vitro of the phytopathogenic fungus Fusarium oxysporum. [ABSTRACT FROM AUTHOR]

Lysosomal proteinases are translated as preproforms, transported through the Golgi apparatus as proforms, and localized in lysosomes as mature forms. In this study, we analyzed which subclass of proteinases participates in the processing of lysosomal proteinases using Bafilomycin Al, a vacuolar ATPase inhibitor. Bafilomycin Al raises lysosomal pH resulting in the degradation of lysosomal proteinases such as cathepsins B, D, and L. Twenty-four hours after the withdrawal of Bafilomycin Al, NIH3T3 cells possess these proteinases in amounts and activities similar to those in cells cultured in DMEM and 5% BCS. In the presence of various proteinase inhibitors, procathepsin processing is disturbed by E-64-d, resulting in abnormal processing of cathepsins D and L, but not by APMSF, Pepstatin A, or CA-074. In the presence of Helicobacter pylori Vac A toxin, which prevents vesicular transport from late endosomes to lysosomes, the processing of procathepsins B and D occurs, while that of procathepsin L does not. Thus, procathepsins B and D are converted to their mature forms in late endosomes, while procathepsin L is processed to the mature form after its arrival in lysosomes by some cysteine proteinase other than cathepsin B. [ABSTRACT FROM PUBLISHER]

Resting seeds of Scots pine (Pinus sylvestris L.) contain inhibitors which inhibit the proteinase activity present in germinating seeds but have no effect on trypsin or chymotrypsin. When a crude inhibitor preparation was chromatographed on Sephadex G-75, the inhibitor activity separated into four peaks with elution volumes corresponding to the molecular weights 24,000, 14,600, 14,000, and 9000. Each of the inhibitors affected both the hydrolysis of haemoglobin at pH 3.7 and the hydrolysis of casein at pH 5.4 and 7.0 by proteinase extracts prepared from "germinating" endosperms. These results suggest that one major proteinase was possibly acting in all the assays. In resting seeds inhibitor activity was present in both the embryo and the endosperm, the activity (per mg dry weight) in the embryo being about 2-fold that in the endosperm. In the endosperms of germinating seeds the inhibitor activity per seed decreased at about the same rate as total N and dry weight. In the seedlings the activity per seedling remained approximately constant. The patterns of the activity changes suggest that the inhibitors do not control the breakdown of storage proteins; a more probable function is the protection of other cellular components from the high proteinase activities required for the rapid proteolysis during germination. [ABSTRACT FROM AUTHOR]

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable, the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7, 90% at pH 5.4, and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 5°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors can be induced by wounding in shoots of tomato (Lycopersicon esculentum [L.] Mill. cv. Moneymaker). These inhibitors are toxic to insects, but their ecological importance is not clear. Published work suggests that proteinase inhibitors may be wound‐inducible in tomato only while the plants are young (less than 30 days). In the present investigation the influence of plant age on wound‐inducible proteinase inhibitor was re‐assessed using tomato plants grown in an outdoor polythene tunnel, with natural lighting and without supplementary heat. In contrast to previous findings, proteinase inhibitor was shown to be induced by wounding in plants of all ages. However, the systemic efficacy of wounds was much reduced in mature plants, possibly because such plants have outgrown the range of the wound‐signalling system. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors (PI) are expressed in several members of the Solanaceae. According to species, PI can be both developmentally and environmentally regulated. We investigated the presence and developmental regulation of PI in the flowers of Nicotiana plumbaginifolia. By trypsin affinity chromatography a small protein, Np‐PI‐II, was isolated. This protein had an N‐terminus similar to a part of a Nicotiana alata PI‐II, and an inhibitory activity towards trypsin. Inhibitory activity is expressed at very early stages of floral development. No inhibitor is present in the vegetative meristems. The inhibitory activity remains high throughout flower maturation and rapidly declines in the ovary after pollination. In the stigma of mature Nicotiana tabacum flowers trypsin inhibitory activity is detected extracellularly. Proteinase inhibitor activity is not a regular feature of the stigmas of several solanaceous genera. Proteinase inhibitors apparently have no function in the development of floral structures and probably act as protective agents against fungal infection of and insect damage to the floral parts and the developing fruit. [ABSTRACT FROM AUTHOR]