Effects of a Peptide Derived from the Primary Sequence of a Kallikrein Inhibitor Isolated from Bauhinia bauhinioides (pep-BbKI) in an Asthma-COPD Overlap (ACO) Model.

(1) There are several patients with asthma-COPD overlap (ACO). A peptide derived from the primary sequence of a kallikrein inhibitor isolated from Bauhinia bauhinioides (pep-BbKI) has potent anti-inflammatory and antioxidant effects. Purpose: To investigate the effects of pep-BbKI treatment in an ACO model and compare them with those of corticosteroids. (2) BALB/c mice were divided into groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep-BbKI (treated with inhibitor), ACO-DX (dexamethasone treatment), ACO-DX-pep-BbKI (both treatments), and SAL-pep-BbKI (saline group treated with inhibitor). We evaluated: hyperresponsiveness to methacholine, bronchoalveolar lavage fluid (BALF), exhaled nitric oxide (eNO), IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TNF-α, MMP-9, MMP-12, TGF-ß, collagen fibers, iNOS, eNO, linear mean intercept (Lm), and NF-κB in airways (AW) and alveolar septa (AS). (3) ACO-pep-BbKI reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, neutrophils, IL-5, IL-10, IL-17, IFN-γ, TNF-α, MMP-12 (AW), collagen fibers, iNOS (AW), and eNO (p > 0.05). ACO-DX reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, total cells and differentials, IL-1ß(AS), IL-5 (AS), IL-6 (AS), IL-10 (AS), IL-13 (AS), IFN-γ, MMP-12 (AS), TGF-ß (AS), collagen fibers (AW), iNOS, and eNO (p > 0.05). SAL was similar to SAL-pep-BbKI for all comparisons (p > 0.05). (4) Pep-BbKI was similar to dexamethasone in reducing the majority of alterations of this ACO model.

Surviving in the fish gut: Comparative inhibitory capacities against the host proteinases in cestodes of the genus Proteocephalus.

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.

Poplar protease inhibitor expression differs in an herbivore specific manner.

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.

Localization of the proteinase inhibitor activity in the fish cestode Eubothrium rugosum.

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.

Association between plasminogen activator inhibitor­1 (PAI-1) 4G/5G polymorphism and circulating PAI-1 level in systemic lupus erythematosus and rheumatoid arthritis : A meta-analysis.

OBJECTIVE: This study systemically reviewed the evidence regarding the association between plasminogen activator inhibitor­1 (PAI­1) 4G/5G polymorphism and susceptibility to systemic lupus erythematous (SLE)/lupus nephritis (LN) and rheumatoid arthritis (RA) and the relationship between circulating PAI­1 levels and SLE/LN and RA. METHODS: We conducted a meta-analysis on the association between the PAI­1 4G/5G polymorphism and SLE/LN or RA risk and serum/plasma PAI­1 levels in patients with SLE/LN and RA and healthy controls. RESULTS: Nine articles including 657 patients with SLE and 668 controls and 567 patients with RA and 772 controls were included. No association was revealed between SLE and PAI­1 4G allele in all study subjects (odds ratio [OR] = 0.944, 95% confidence interval [CI] = 0.808-1.102, p = 0.463). Ethnicity-based stratification showed no association between the PAI­1 4G allele and SLE among Europeans and Asians. No association was detected between LN and RA and the PAI­1 4G allele (OR = 0.886, 95% CI = 0.713-1.102, p = 0.278; OR = 0.8736, 95% CI = 0.747-1.020, p = 0.088, respectively) or between SLE/LN and RA and the PAI­1 4G/5G polymorphism using the recessive and dominant models and homozygote contrast. The circulating PAI­1 level was significantly higher in the SLE group than in the control group (standardized mean difference [SMD] = 0.337, 95% CI = 0.057-0.619, p = 0.019). However, serum/plasma PAI­1 level showed no significant difference between RA and control group (SMD = 0.333, 95% CI = -0.6989-1.35, p = 0.527). CONCLUSIONS: There was no association between the PAI­1 4G/5G polymorphism and SLE/LN and RA development and significantly higher levels of circulating PAI­1 were observed in patients with SLE but not in those with RA.

A Kunitz proteinase inhibitor (HcKuPI) participated in antimicrobial process during pearl sac formation and induced the overgrowth of calcium carbonate in Hyriopsis cumingii.

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.

Purification and characterization of two cysteine proteinase inhibitors from silkworm, Bombyx mori.

Cysteine proteinase inhibitors from silkworm are selective inhibitors with low molecular weight and regulate cathepsin L-like cysteine proteinase activity, thus, affecting silkworm metamorphosis. In a previous study, two cysteine proteinase inhibitors, BCPI and BmCPI, were identified in the silkworm genome. To characterize these inhibitors, we expressed and purified them in an Escherichia coli system and analyzed their structure and inhibitory activity in vitro. Both inhibitors showed strong tolerance to high temperature. Their CD spectra revealed that their secondary structures could be recovered by a gradual decrease in temperature. Compared to BCPI, BmCPI exhibited weak inhibitory activity toward cathepsin L. BCPI activity was significantly decreased when its C-terminus was truncated, whereas BmCPI activity increased considerably when the C-terminus tail of BCPI was attached to BmCPI. Additionally, the inhibitory activity of BCPI was strongly reduced if R31 was mutated to A31. In summary, two cysteine proteinase inhibitors from silkworm were characterized in the present study, which facilitates an understanding of the interaction mechanism between cysteine proteinase and its inhibitors in the silkworm.

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

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.

Alpha-1 antitrypsin treatment of new-onset type 1 diabetes: An open-label, phase I clinical trial (RETAIN) to assess safety and pharmacokinetics.

OBJECTIVE: To determine the safety and pharmacokinetics of alpha-1 antitrypsin (AAT) in adults and children. RESEARCH DESIGN AND METHODS: Short-term AAT treatment restores euglycemia in the non-obese mouse model of type 1 diabetes. A phase I multicenter study in 16 subjects with new-onset type 1 diabetes studied the safety and pharmacokinetics of Aralast NP (AAT). This open-label, dose-escalation study enrolled 8 adults aged 16 to 35 years and 8 children aged 8 to 15 years within 100 days of diagnosis, to receive 12 infusions of AAT: a low dose of 45 mg/kg weekly for 6 weeks, followed by a higher dose of 90 mg/kg for 6 weeks. RESULTS: C-peptide secretion during a mixed meal, hemoglobin A1c (HbA1c), and insulin usage remained relatively stable during the treatment period. At 72 hours after infusion of 90 mg/kg, mean levels of AAT fell below 2.0 g/L for 7 of 15 subjects. To identify a plasma level of AAT likely to be therapeutic, pharmacodynamic ex vivo assays were performed on fresh whole blood from adult subjects. Polymerase chain reaction (PCR) analyses were performed on inhibitor of IKBKE, NOD1, TLR1, and TRAD gene expression, which are important for activation of nuclear factor-κB (NF-κB) and apoptosis pathways. AAT suppressed expression dose-dependently; 50% inhibition was achieved in the 2.5 to 5.0 mg/mL range. CONCLUSIONS: AAT was well tolerated and safe in subjects with new-onset type 1 diabetes. Weekly doses of AAT greater than 90 mg/kg may be necessary for an optimal therapeutic effect.

[Changes of Nonspecific Proteinases and Proinflammatory Cytokines in the Clinical Course of Acute Myocardial Infarction of Various Severity].

PURPOSE: to study changes of serum levels of nonspecific proteinases, their inhibitors, and proinflammatory cytokines during short term observation of patients with acute myocardial infarction (MI). MATERIALS AND METHODS: We included in this prospective short-term study 82 patients (27 with uncomplicated non-Q wave MI, 30 with Q-MI complicated by Killip class I-II acute left ventricular failure [ALVF], 17 with Q-MI complicated by Killip class III-IV ALVF, and 8 non-survivors due to development of cardiogenic shock) and 12 healthy controls. Serum levels of interleukin (IL) - 1ß, IL-6 and tumor necrosis factor (TNF)-α were measured by ELISA. Elastaselike (ELA) and trypsin-like (TLA) activities as well as characteristics of proteinase inhibitors (antitrypsin activity and acid-stable inhibitors) were also determined. Blood samples were taken at hospital admission within 24 hours after onset of symptoms. The GUSTO Score at admission was used for risk stratification. RESULTS: All cytokines levels were significantly elevated in MI patients in comparison to controls. Mean concentrations of IL-6 and TNF-α at baseline were higher among patients with MI complicated by ALVF than in the group with uncomplicated MI (27.45 vs 16.04 pikogram / mL, р.

The genome of Strongyloides spp. gives insights into protein families with a putative role in nematode parasitism.

Parasitic nematodes are important and abundant parasites adapted to live a parasitic lifestyle, with these adaptations all aimed at facilitating their survival and reproduction in their hosts. The recently sequenced genomes of four Strongyloides species, gastrointestinal parasites of humans and other animals, alongside transcriptomic and proteomic analysis of free-living and parasitic stages of their life cycles have revealed a number of protein families with a putative role in their parasitism. Many of these protein families have also been associated with parasitism in other parasitic nematode species, suggesting that these proteins may play a fundamental role in nematode parasitism more generally. Here, we review key protein families that have a putative role in Strongyloides' parasitism - acetylcholinesterases, astacins, aspartic proteases, prolyl oligopeptidases, proteinase inhibitors (trypsin inhibitors and cystatins), SCP/TAPS and transthyretin-like proteins - and the evidence for their key, yet diverse, roles in the parasitic lifestyle.

Antioxidant and Proteinase Inhibitory Activities of Selected Poppy (Papaver somniferum L.) Genotypes.

Poppy seeds (Papaver somniferum L.) belong to tasty food ingredients however, they should be considered also as valuable source of biologically active compounds. Content of selected metabolites, antioxidant and proteinase inhibitory activities were analyzed in vitro in extracts from seeds of fifteen poppy genotypes. Considerable variation in all parameters was detected within the set of analyzed poppy genotypes. The genotype Major expressed the highest antioxidant activity determined by all four methodological approaches (DPPH, ABTS, FRAP, RP). The genotype MS 423 exhibited the highest inhibitory activities against trypsin, thrombin and collagenase. Very specific position among all had the genotype Redy. Its grain extract reached significantly high levels in 9 out of 14 measured parameters (TPC, TFC, TTC, TAC, FRAP, RP, inhibitory activities against trypsin, thrombin, collagenase). Edible grains of poppy are valuable source of natural compounds which may be beneficial in pathological states associated with oxidative stress or increased proteinase activities.

Herbivores with similar feeding modes interact through the induction of different plant responses.

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.

Down-regulation of plant defence in a resident spider mite species and its effect upon con- and heterospecifics.

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.

The Potential Role of Kallistatin in the Development of Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a vascular condition that causes permanent dilation of the abdominal aorta, which can lead to death due to aortic rupture. The only treatment for AAA is surgical repair, and there is no current drug treatment for AAA. Aortic inflammation, vascular smooth muscle cell apoptosis, angiogenesis, oxidative stress and vascular remodeling are implicated in AAA pathogenesis. Kallistatin is a serine proteinase inhibitor, which has been shown to have a variety of functions, potentially relevant in AAA pathogenesis. Kallistatin has been reported to have inhibitory effects on tumor necrosis factor alpha (TNF-α) signaling induced oxidative stress and apoptosis. Kallistatin also inhibits vascular endothelial growth factor (VEGF) and Wnt canonical signaling, which promote inflammation, angiogenesis, and vascular remodeling in various pre-clinical experimental models. This review explores the potential protective role of kallistatin in AAA pathogenesis.

Molecular insights into mechanisms of lepidopteran serine proteinase resistance to natural plant defenses.

Plants have a wide range of chemical defenses against predation, including substances that target digestive serine proteinases of herbivorous. Previous works demonstrated that lepidopteran insects have digestive serine proteinases resistant to plant proteinase inhibitors (PPIs) and ketone modifications, while coleopteran ones are sensitive to those plant defenses. This paper focuses on molecular aspects that lead lepidopteran serine proteinases to PPI and ketone modification resistance. Using biochemical experiments and computer 3D modeling we demonstrated that lepidopteran trypsins are more hydrophobic than coleopteran ones, a feature associated to trypsin oligomerization and decreased inhibition by PPI. Moreover, the determination of pKa values of chymotrypsin catalytic residues obtained by TPCK modification indicates that the environment around the active site of ketone-resistant and -sensitive chymotrypsins are different. Structural analysis using resistant and sensitive chymotrypsins data allowed us to point 2 hotspot regions around the active site that could explain the observed differences. Our set of results highlights features of serine proteinases important for understanding the resistance of insects to plant chemical defenses.

Fluorescent analogs of trypsin inhibitor SFTI-1 isolated from sunflower seeds--synthesis and applications.

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'-trimethoxyphenyl)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 [Phe(5)]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.

Maternal serum serpin B7 is associated with early spontaneous preterm birth.

OBJECTIVE: We sought to identify serum biomarkers of early spontaneous preterm birth (SPTB) using semiquantitative proteomic analyses. STUDY DESIGN: This was a nested case-control study of pregnant women with previous SPTB. Maternal serum was collected at 19-24 and 28-32 weeks' gestation, and analyzed by liquid chromatography-multiple reaction monitoring/mass spectrometry. Targeted and shotgun proteomics identified 31 candidate proteins that were differentially expressed in pooled serum samples from spontaneous preterm (cases [<34 weeks]) and term (controls) deliveries. Candidate protein expression was compared in individual serum samples between cases and controls matched by age and race groups, and clinical site. Protein expression was verified by Western blot in the placenta and fetal membranes from cases and controls. RESULTS: Serum samples were available for 35 cases and 35 controls at 19-24 weeks, and 16 cases and 16 controls at 28-32 weeks. One protein, serpin B7, yielded serum concentrations that differed between cases and controls. The mean concentration of serpin B7 at 28-32 weeks was 1.5-fold higher in women with subsequent preterm deliveries compared to controls; there was no difference at 19-24 weeks. Higher levels of serpin B7 at both gestational age windows were associated with a shorter interval to delivery, and higher levels of serpin B7 in samples from 28-32 weeks were associated with a lower gestational age at delivery. Western blotting identified serpin B7 protein in placenta, amnion, and chorion from cases and controls. CONCLUSION: Targeted and shotgun serum proteomics analyses associated 1 protein, serpin B7, with early SPTB. Our results require validation in other cohorts and analysis of the possible mechanistic role of serpin B7 in parturition.

Molecular cloning of α-2-macroglobulin from hemocytes of common periwinkle Littorina littorea.

We report the sequence of the proteinase inhibitor with a wide inhibition spectrum, α-2-macroglobulin (α2M), belonging to the thioester superfamily of proteins. This is the first α2M sequence from coenogastropod prosobranch snails. The full-length cDNA was cloned by RACE method, spans 7897 bp and contains an open reading frame of 5460 bp. The ORF encodes a protein of 1819 amino acids. The deduced mature protein contains 1795 amino acids with a molecular weight of 200 kDa and isoelectric point of 5.00. Littorina littorea α2M bears 4 conserved α2M domains and one internal thioester. Phylogenetic analysis showed that the sequence forms well supported cluster with Mollusca species and other representatives of Lophotrochozoa.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

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.