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Kazal-type serine proteinase inhibitors (KPIs) function in physiological and immunological processes requiring proteinase action. In the present study, the first Cherax quadricarinatus KPI gene (designated CqKPI) was identified and characterized. The open reading frame of CqKPI contains 405 nucleotides and encodes a protein of 134 amino acids. CqKPI has two Kazal domains comprising 44 amino acid residues with the conserved amino acid sequence C-X 3 -C-X 7 -C-X 6 -Y-X 3 -C-X 6 -C-X 12 -C. Each Kazal domain has six conserved cysteine residues, which can form a structural conformation of three pairs of disulfide bonds stabilizing the Kazal domain. CqKPI exhibited high similarity with previously identified KPIs from crayfish hemocytes. The results of tissue distribution showed that CqKPI had the highest expression level in hemocytes, and this was in agreement with phylogenic relationships. Recombinant CqKPI (rCqKPI) was heterologously expressed in Escherichia coli and purified for further study. The proteinase inhibition assays suggested that rCqKPI could potently inhibit elastase and weakly inhibit trypsin, subtilisin A, and proteinase K, but not α-chymotrypsin. It can firmly bind to Bacillus hwajinpoensis, Staphylococcus aureus, and Vibrio parahaemolyticus, with weak binding to Candida albicans. In addition, CqKPI inhibited bacterial secretory proteinase activity and inhibited the growth of B. hwajinpoensis and C. albicans. These data suggest that CqKPI might be involved in anti-bacterial immunity, acting as an inhibitor of the proteinase cascade in the resistance to invasion of pathogens., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We describe the total synthesis of tutuilamide A, a potent porcine pancreatic elastase (PPE) inhibitor and a representative member of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide family, isolated from marine cyanobacteria. The Ahp unit serves as a pharmacophore and the adjacent 2-amino-2-butenoic acid (Abu) is a main driver of the selectivity among serine proteases. We adapted our previous convergent strategy to generate the macrocycle, common with lyngbyastatin 7 and related elastase inhibitors, and then appended the tutuilamide A-specific side chain bearing a vinyl chloride. Tutuilamide A and lyngbyastatin 7 were evaluated side by side for the inhibition of the disease-relevant human neutrophil elastase (HNE). Tutuilamide A and lyngbyastatin 7 were approximately equipotent against HNE, while tutuilamide A was previously shown to be more active against PPE compared with lyngbyastatin 7, further demonstrating that the side chain provides opportunities to not only modulate potency but also selectivity among proteases of the same function from different organisms. Profiling of tutuilamide A against mainly human serine proteases revealed high selectivity for HNE (IC 50 0.73 nM) and pleiotropic activity against kallikrein 7 (KLK7, IC 50 5.0 nM), without affecting other kallikreins, similarly to lyngbyastatin 7 (IC 50 0.85 nM for HNE and 3.1 nM for KLK7). A comprehensive molecular docking study for elastases and KLK7 afforded deeper insight into the intricate differences between inhibitor interactions with HNE and PPE, accounting for the differential activities for both compounds. The synthesis and molecular studies serve as a proof-of-concept that the macrocyclic scaffold can be diversified to fine-tune the activity of serine protease inhibitors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa's active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease (AHPND) is a recently emerged disease in aqua cultured shrimp that is caused by virulent strains of Vibrio parahaemolyticus (VP). Our previous study used transcriptomics to identify key pathogenic factors in the stomach of AHPND-infected shrimp (Litopenaeus vannamei), and here we used a different subset of the same data to construct a gene-to-gene expression correlation network to identify immune-responsive genes. LvSerpin7 was found to have the highest number of correlations after infection, and it also showed a significant increase in mRNA expression. LvSerpin7 is expressed in all tissues but its expression levels are highest in hemocytes. After successfully silencing LvSerpin7 transcript prior to AHPND challenge, mortality was significantly increased relative to the controls and reached 100% within 36 h post infection. Compared to the controls, the phenoloxidase (PO) activity also increased in both hemolymph and stomach. Recombinant LvSerpin7 inhibited shrimp PO activity in vitro, and we also found that rLvSerpin7 inhibited the growth of AHPND-causing bacteria. These results suggest that LvSerpin7 might reduce the toxic effects that result from unregulated activation of the PO defense system by AHPND-causing bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Serine protease inhibitors (SPIs) play an important role in cell survival, development and host defense. In plants, serine protease inhibitors such as the Kunitz-type inhibitor (KTI) and the Bowman-Birk inhibitor (BBI) have been shown to be induced in response to abiotic stress such as salinity and drought resulting in tolerance to these stresses. In this study, Arabidopsis thaliana (T3) plants overexpressing the BBI gene from maize were generated and subjected to drought stress in order to study the role of BBI protease inhibitor in drought tolerance. Drought treatment of four-week-old Arabidopsis plants was performed by withholding water from plants for nine days and harvested plant material was used for physiological and biochemical analysis. The transgenic lines exhibited normal growth after nine days of drought as compared to the wild-type. The results also showed a higher leaf relative water content (RWC) in transgenic lines when compared to the wild-type (WT), with line 2 having the highest RWC of 72% and the WT having the lowest RWC of 32%. Trypsin-inhibitor activity indicated that the total protein of the positive transgenic plants had stronger protease inhibitory activity than the wild-type. Transgenic lines overexpressing BBI also showed reduced lipid peroxidation (MDA content) as well as enhanced activity of antioxidants glutathione-s-transferase (GST) and ascorbate peroxidase (APX). These results suggest that BBI protease inhibitor leads to drought tolerance associated with reduction in drought-induced oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The protease inhibitors found in silkworm cocoons can be divided into several families, a majority of which contain serpin, TIL, or Kunitz domains. Previously, it has been reported that TIL-type protease inhibitors have antimicrobial activity. To date, however, it has not been determined whether the Kunitz-type protease inhibitor BmSPI51, the most abundant of cocoon protease inhibitors, plays an antimicrobial role. Thus, in this study, we sought to determine the biological role of BmSPI51 in silkworm cocoons. Our results obtained from real-time quantitative reverse transcription PCR and immunofluorescence analyses indicate that BmSPI51 is expressed exclusively in the silk glands during the larval fifth instar stage and is subsequently secreted into cocoon silk. Moreover, at a molar ratio of 1:1, BmSPI51 produced via prokaryotic expression exhibited inhibitory activity against trypsin and also proved to be highly stable over wide ranges of temperature and pH values. The expression of BmSPI51 was also found to be significantly upregulated in the larval fat body after infection with three species of fungi, namely, Candida albicans, Beauveria bassiana, and Saccharomyces cerevisiae. In vitro inhibition tests revealed that BmSPI51 significantly inhibited the sporular growth of all three of these fungal species. Further, results obtained from a binding assay showed that BmSPI51 binds to β-d-glucan and mannan on the surface of fungal cells. In this study, we, thus, revealed the antimicrobial activity of BmSPI51 and its underlying mechanism in silkworm, thereby contributing to our present understanding of defense mechanisms in silkworm cocoons., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Some serine protease inhibitor (serpin) regulators of essential life pathways bind glycosaminoglycans (GAGs) to enhance inhibitory functions and achieve physiologically relevant rates. This study demonstrates that highly conserved Amblyomma americanum tick saliva serpin 19 (AAS19), a broad-spectrum inhibitor of hemostasis and inflammation system proteases and anticoagulant, can bind heparan sulfate/heparin (HS)GAGs and that this interaction alters its function. Substrate hydrolysis and unpaired t-test analyses revealed that HSGAG binding caused rAAS19 inhibitory activity to: (i) significantly increase against blood clotting factors (f) IIa (thrombin) and fIXa, (ii) significantly reduce against fXa and fXIIa and (iii) moderate to no effect against trypsin, kallikrein, papain, and plasmin. Stoichiometry of inhibition (SI) analyses show that HSGAG binding improved the rAAS19 inhibitory efficiency against thrombin 2.7-4.3 fold as revealed by SI change from 13.19 in absence of HSGAGs to 4.83-3.04 in their presence. Our data show that HSGAG binding dramatically enhanced rAAS19 anticoagulant function. In the recalcification time assay, rAAS19 pre-incubated with HSGAGs prior to the assay, delayed plasma clotting by an additional 176-457 s above HSGAGs or rAAS19 alone. Our data suggest that formation of the HSGAGs and rAAS19 complex is important for the observed enhanced anticoagulant effect. Delay of plasma clotting was higher when HSGAGs and rAAS19 were co-incubated to allow complex formation prior to blood clotting assay as opposed to no co-incubation. We have discussed our finding with reference to tick feeding physiology and utility of the rAAS19 in blood clotting disorder therapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Serine protease inhibitors (SPIs) play a key role in physiological responses by controlling protease activities. In this study, we studied the biochemical functions of serpin-15, an SPI, from Bombyx mori (Bmserpin-15). Recombinant Bmserpin-15 was expressed in Escherichia coli cells and used to raise rabbit anti-Bmserpin-15 polyclonal antibodies. Bmserpin-15 mRNA and protein expression was detected in all tested tissues, particularly in the fat body and silk gland. After challenge with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus and B. mori nuclear polyhedrosis virus), the expressions of Bmserpin-15 mRNA and protein were induced significantly, particularly by B. bassiana and M. luteus. Recombinant Bmserpin-15 inhibited prophenoloxidase activation, but did not affect phenoloxidase activity, in B. mori hemolymph. Injection of recombinant Bmserpin-15 into B. mori larvae reduced significantly the transcript levels of antimicrobial peptides in fat body. Our results suggested that Bmserpin-15 plays an important role in the innate immunity of B. mori., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Serine protease inhibitors (Serpins) are a large family of protease inhibitors involved in many critical biological processes such as blood coagulation, fibrinolysis, programmed cell death, development, and innate immunity. We identified MjSerp1, a serpin in the kuruma shrimp Marsupenaeus japonicus. The MjSerp1 cDNA has a 1239 bp open reading frame (ORF) that encodes a 412-amino acid protein with a 23 aa signal peptide and a classic serpin domain. MjSerp1 has a calculated molecular mass of 46.3 kDa and a predicted isoelectric point of 5.51. MjSerp1 is mainly expressed in the hepatopancreas and the intestines, and is moderately expressed in hemocytes. Expression pattern analysis indicated that MjSerp1 is upregulated in the hepatopancreas after Vibrio anguillarum challenge. rMjSerp1 inhibits three Gram-positive bacteria and two Gram-negative bacteria, but does not inhibit phenoloxidase activity. The microorganism binding assay showed that rMjSerp1 closely binds to both Gram-positive and Gram-negative bacteria. MjSerp1 also exhibits inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, indicating that MjSerp1 acts as a microbial serine protease inhibitor. rMjSerp1 injection into shrimp enhances V. anguillarum clearance, but MjSerp1 knockdown through RNA interference impairs Vibrio clearance in vivo. These results indicate that MjSerp1 functions as a direct effector in the bacterial clearance of M. japonicus. All together, our findings provide novel evidences for the serine protease inhibitor in shrimp immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

As important arthropod immune responses, prophenoloxidase (proPO) activation and Toll pathway initiation are mediated by serine proteinase cascades and regulated by serpins. Herein, a serine protease inhibitor (Lvserpin), encoding for 415 amino acids with calculated molecular weight of 46,639 Da and isoelectric point of 7.03 was characterized from the Pacific white shrimp Litopenaeus vannamei. Multiple sequence alignment revealed that Lvserpin shared the highest similarity with Penaeus monodon serpin6 (87%). Quantitative real-time PCR (qRT-PCR) results showed that the transcripts of Lvserpin were detected in all the examined tissues and most highly expressed in gill. The expression profiles of Lvserpin were greatly fluctuated upon infection of Vibrio anguillarum, Micrococcus lysoleikticus or White Spot Syndrome Virus (WSSV). Double stranded RNA-mediated suppression of Lvserpin resulted in a significant increase in the transcripts of two clip-domain serine proteinases (PPAE and PPAF), prophenoloxidase (proPO), anti-lipopolysaccharide factor (ALF), Crustin and penaeidin3 (Pens3) and also increased the high cumulative mortality post V. anguillarum injection. Besides, the recombinant Lvserpin protein (rLvserpin) was purified and exhibited inhibitory activity against trypsin. Also the rLvserpin showed inhibition on prophenoloxidase activation and bacterial growth. Hence, we proposed that the Lvserpin played important role in the shrimp innate immunity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mosquito saliva carries a large number of factors with anti-hemostatic, anti-inflammatory and immuno-modulatory activities. The cE5 protein was initially identified during an Anopheles gambiae salivary gland transcriptome study and later shown to share sequence similarity with anophelin, a thrombin inhibitor from the saliva of the New World mosquito Anopheles albimanus. The cE5 gene was found to encode different mRNA isoforms coexisting in several tissues of both male and female mosquitoes, a highly unusual profile for a gene potentially encoding an anti-thrombin and involved in blood feeding. Expression of the cE5 protein and assessment of its activity and inhibitory properties showed that it is a highly specific and tight-binding thrombin inhibitor, which differs from the A. albimanus orthologue for the fast-binding kinetics. Despite the widespread occurrence of cE5 transcripts in different mosquito tissues the corresponding protein was only found in female salivary glands, where it undergoes post-translational modification. Therefore, tissue-specific restriction of the A. gambiae cE5 is not achieved by transcriptional control, as common for mosquito salivary genes involved in hematophagy, but by post-trascriptional gene regulatory mechanisms. Our observations provide a paradigm of post-transcriptional regulation as key determinant of tissue specificity for a protein from an important disease vector and point out that transcriptomic data should be interpreted with caution in the absence of concomitant proteomic support., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor-activated factor VII complex. In addition, TFPI-2 has other functions such as promoting cell migration and inducing apoptosis. In the present study, we investigated if TFPI-2 induced apoptosis in cultured U937-derived macrophages and the possible signal pathways that involved in the apoptotic process. Apoptotic DNA fragment detection and caspase-3,9 activity measurements indicated that rTFPI-2 promoted U937-derived macrophage apoptosis. Hoechst 33342 assay and flow cytometry further showed that rTFPI-2 induced apoptosis in cultured macrophages in a dose-dependent manner. Because death receptors of the TNF family such as Fas are the best-understood death pathways that recruit Fas-associated death domain (FADD) and procaspase-8 to the receptor in macrophages, we investigated the expression of Fas and its ligand (FasL) and downstream signal caspase-8 by Western blot analysis. The results indicated that the process of apoptosis triggered by rTFPI-2 was, at least in part, actively conducted by U937-derived macrophages possibly through Fas/FasL signal pathway. In brief, rTFPI-2 may have the potential usefulness in inducing macrophages apoptosis, which suggest TFPI-2 might have antiatherogenic effects., (Copyright © 2010 Elsevier B.V. All rights reserved.)

An assay to quantify bradykinin in rat plasma has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sar-D-Phe(8)-des-Arg(9)-bradykinin was used as internal standard. Aprotinin was added to rat plasma to inhibit the activity of proteinases. Recoveries for solid-phase extraction (SPE) on Strata X reversed phase were greater than 80%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with an electrospray source (ESI), operating in the positive ion-mode, was used for detection. The assay was validated and stability was explored. Bradykinin (10-500 ng/mL) was quantified with accuracy values (% RE) below 10% and intra- and inter-day precisions (% RSD) below 12 and 16%, respectively, for all concentrations. The method was successfully applied to several plasma samples from low levels kallikrein rats (LKRs) compared with normal kallikrein rats (NKRs)., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We characterized a Kunitz-type protease inhibitor (Gm KTPI) obtained from the hemolymph of Galleria mellonella larvae immunized with Escherichia coli. The structural analysis of the cloned cDNA showed that it consists of 56 residues derived from the precursor of 75 amino acids. The peptide was constitutively produced in the fat bodies, but not in the midgut nor the integument of larvae. In our analysis of stage-dependent expression, its transcript was detected within the midgut, the fat bodies and the integument of the prepupae, which undergo tissue remodeling. The inhibition assays showed that Gm KTPI was capable of inhibiting only the trypsin-like activity of the larval midgut extracts. Furthermore, it was determined that Gm KTPI induced the activation of extracellular signal-regulated kinase (ERK) in the fat bodies and integument cells, and this kinase is known to perform a central role in cell proliferation signaling. Its effect on ERK activation was also verified in a control experiment using a human endothelial cell culture. Collectively, it was suggested that Gm KTPI might be responsible for the protection of other tissues against proteolytic attack by trypsin-like protease(s) from larval midgut during metamorphosis, and might play a role in the proliferation of cells in the fat body and integument., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Suppression subtractive hybridization (SSH) was employed to identify yellow head virus (YHV)-responsive genes from the hemocytes of the black tiger shrimp, Penaeus monodon. Two SSH cDNA libraries were constructed to identify viral responsive genes in the early (24I) and late (48/72I) phases of YHV infection. From 240 randomly selected clones from each library, 155 and 30 non-redundant transcripts were obtained for the early and late libraries, respectively. From these clones, 72 and 16, respectively, corresponded to known genes (E-values < 1 x 10(-4)) that could be categorized according to their putative functions. The upregulated genes identified as likely to be associated with cell defense and homeostasis were found at a high proportion in the 24I SSH library, but not in 48/72I SSH library implying that these immune molecules participate in viral defense immunity in the early phase of YHV infection whereas their expressions were suppressed in the late phase of infection. Novel YHV-responsive genes were uncovered from these SSH libraries including caspases, histidine triad nucleotide-binding protein 2, Rab11, beta-integrin, tetraspanin, prostaglandin E synthase, transglutaminase, Kazal-type serine proteinase inhibitor and antimicrobial peptides. Among these YHV-responsive genes, several have been previously reported to participate in defense against white-spot syndrome virus (WSSV) implying that YHV infection in shrimp induces similar host immune responses as observed during WSSV infection. The expression of four apparently upregulated immune-related genes identified from the two SSH libraries, anti-lipopolysaccharide factor isoform 6 (ALFPm6), crustin isoform 1 (crustinPm1), transglutaminase and Kazal-type serine proteinase inhibitor isoform 2 (SPIPm2), was evaluated by real-time RT-PCR to reveal differential expression in response to YHV infection at 6, 24, 48 and 72 h post-infection. The results confirmed their differential expression and upregulation, and thus verified the success of the SSHs and the likely involvement of these genes in shrimp antiviral mechanisms., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Triatomines inhibit the clotting of ingested blood in the stomach (anterior midgut). After verifying this phenomenon in Panstrongylus megistus using coagulation assays, a full-length cDNA encoding a Kazal-like inhibitor was amplified by PCR. The open reading frame encodes a putative precursor protein of 412 amino acid residues, which was named PmStKaz and contains seven Kazal-like domains forming four Kazal-type inhibitors. A single domain inhibitor and three double-domain inhibitors possess sequence identities of up to 91% to the respective domains of Kazal-type inhibitors from other triatomines. The gene is expressed in the stomach (anterior midgut) but not in the small intestine (posterior midgut), salivary glands or haemocytes. After hydrophobic interaction chromatography of the stomach contents, four fractions (numbers 1-4) inhibited the activity of trypsin, fraction 2 that of subtilisin A, fractions 1, 3 and 4 that of plasmin, and fractions 3 and 4 that of thrombin. After ion exchange chromatography, MALDI-TOF-MS analysis of the intact proteins in fractions 3 and 4 showed diverse masses correlating to PmStKaz IV-V and PmStKaz II-III, respectively. Both proteins seem to be present in several isoforms with variant amino- and carboxy-terminal ends. In reverse zymography of the proteins of the stomach contents after separation by isoelectric focusing and non-reducing SDS-PAGE, much higher concentrations of isoforms of PmStKaz II-III and IV-V were evident than of PmStKaz I and VI-VII., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.

The serine protease inhibitor cvSI-1, purified from plasma of eastern oysters, inhibited the proliferation of the protozoan parasite Perkinsus marinus in vitro. In situ hybridization located cvSI-1 gene expression in basophil cells of the digestive tubules and cvSI-1 expression measured by real-time quantitative reverse transcriptase polymerase chain reaction was several hundred folds greater in digestive glands than in other organs examined or circulating hemocytes. cvSI-1 gene expression was also significantly greater in winter than in summer. Finally, cvSI-1 gene expression and plasma protease inhibitory activity in oysters selected for increased resistance to P. marinus were significantly greater than in unselected oysters. These findings support the hypothesis that cvSI-1 plays a role in eastern oyster host defense against P. marinus possibly through inhibition of parasite proliferation.

In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not alpha-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.

We investigated fibrin-mediated gene transfer by embedding pDNA within the hydrogel during polymerization and using two modes of gene transfection with cells placed either on the surface (2D transfection) or within the hydrogel (3D transfection). Using this model, we found that cell transfection depended strongly on the local cell-pDNA microenvironment as defined by the 2D vs. 3D context, target cell type and density, as well as fibrinogen and pDNA concentrations. When cells were embedded within the fibrin matrix lipofectamine-induced cell death decreased significantly, especially at low target cell density. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner, suggesting that fibrin degradation may be necessary for efficient gene transfer. We also provided proof-of-concept that fibrin-mediated gene transfer can be used for spatially localized gene delivery, which is required in cell-transfection microarrays. When lipoplex-containing hydrogels were spotted in an array format gene transfer was strictly confined to pDNA-containing fibrin spots with no cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may be used as a biomaterial to deliver genes in an efficient, cell-controlled and spatially localized manner for potential applications in vitro or in vivo.

In the absence of migratory phagocytic cells the basal metazoan Hydra has developed a very effective immune system. Previous work has shown that epithelial cells, both in the ectoderm and endoderm, recognize PAMPs by TLR and produce a number of antimicrobial peptides. In this study we demonstrate that not only epithelial cells but also gland cells are critically involved in Hydra's innate host defense by producing a kazal-type serine protease inhibitor, kazal2, that has potent in vitro bactericidal activity against Staphylococcus aureus. The discovery of an antimicrobial serine protease inhibitor in Hydra may shed new light on the mechanisms of host defense early in metazoan evolution, and promises to open new avenues for the development of potent anti-staphylococcal compounds.

Single WAP domain (SWD)-containing proteins are small proteins with a C-terminal region containing a single whey acidic protein (WAP) domain. In the present study, the cDNAs representing three isoforms of SWD proteins (SWDPm1, SWDPm2 and SWDPm3) were identified from hemocytes of the black tiger shrimp, Penaeus monodon. The deduced peptides revealed that they contain a putative signal peptide of 24 amino acids and encode for a mature peptide of 69, 68 and 56 amino acids, respectively, which contain typical characters similar to those of the shrimp SWD proteins (type III crustin) with a Pro-Arg region and a WAP domain towards the C-terminus. Tissue distribution analysis by RT-PCR showed that all three SWDPm transcripts were primarily found in hemocytes. Transcript expression of SWDPm1 was down-regulated upon injection with Staphylococcus aureus whilst there was no change of SWDPm2 and SWDPm3 expression. In contrast, white spot syndrome virus (WSSV) injection resulted in a biphasic response with up-regulation of SWDPm1 and SWDPm2 transcripts at 6h followed by significant down-regulation by 24h after infection. Genomic organization of the SWDPm2 gene revealed the presence of three exons interrupted by two introns. To characterize the biological functions of the SWD protein, the mature SWDPm2 protein encoding cDNA was cloned and expressed in Escherichia coli. Purified recombinant (r)SWDPm2 exhibits antibacterial activity against several Gram-positive, but not Gram-negative, bacteria and is a competitive inhibitor of subtilisin A with an inhibition constant (Ki) of 1.98nM. Thus, rSWDPm2 may contribute to the inhibitory regulation of subtilisin A from bacterial infection and P. monodon SWD protein likely function as immune effectors in defense against invasion of shrimp pathogens.

By an analysis of PDB crystal structures, the mean conformations of protein strands bound in serine protease active sites are shown to contain extensively aligned atomic orbitals. The active-serine-bearing segment of each enzyme (subtilisin BPN' and beta-trypsin) also contains such alignments. The participating orbitals are almost identical in each system. All of the alignments converge on the targeted linkage. They suggest that a kind of through-strand polarizability is being optimized by evolution, presumably due to corresponding benefits in proteolysis rate. Such polarizability would help to explain the high values of kcat seen for long oligopeptide substrates. The idea predicts long substrates to be relatively reactive even under non-enzymatic conditions, which in fact they are.

Thrombin-like enzymes isolated from snake venoms comprise a group of serine proteinases responsible for many important coagulation disorders in the envenomed victims. Besides, these proteinases have great biotechnological interest as antithrombotic agents and as diagnostic tools. However, in spite of the recent overflow of snake venom thrombin-like enzymes (SVTLEs) on protein sequence databases, there is a lack of three-dimensional (3D) structural information on this family. Without such 3D structures available many aspects of the biological function and biochemical properties of these enzymes still remain obscure. Therefore, we have gone through a series of computational techniques, which enabled us to identify the set of residues involved in molecular recognition of inhibitors bound to the S1 subsite of snake venom thrombin-like enzymes (SVTLEs) and ultimately conclude that nonpolar (van der Waals) intermolecular interactions and ligand's hydrophobicity are the most important factors affecting binding affinities to the S1 subsite of a SVTLE isolated from the venom of Lachesis muta muta (Lmm-TLE). Consequently, we have proposed that S1 subsite lipophilicity may be used to sort binding affinities of trypsin-like enzymes to small molecules by showing that the inhibitory potency of several S1-directed compounds follows subsite lipophilicity among Lmm-TLE and other three homologous proteases. Noteworthy, in the course of our analyses we determined that thrombin's S1 subsite should, in fact, be considered less lipophilic than that of trypsin if we account for the presence of the sodium-controlled water channel communicating with the S1 subsite in the coagulant enzyme.

EP and GPEET procyclin, the major surface glycoproteins of procyclic forms of Trypanosoma brucei, are truncated by proteases in the midgut of the tsetse fly Glossina morsitans morsitans. We show that soluble extracts from the midguts of teneral flies contain trypsin-like enzymes that cleave the N-terminal domains from living culture-derived parasites. The same extract shows little activity against a variant surface glycoprotein on living bloodstream form T. brucei (MITat 1.2) and none against glutamic acid/alanine-rich protein, a major surface glycoprotein of Trypanosoma congolense insect forms although both these proteins contain potential trypsin cleavage sites. Gel filtration of tsetse midgut extract revealed three peaks of tryptic activity against procyclins. Trypsin alone would be sufficient to account for the cleavage of GPEET at a single arginine residue in the fly. In contrast, the processing of EP at multiple sites would require additional enzymes that might only be induced or activated during feeding or infection. Unexpectedly, the pH optima for both the procyclin cleavage reaction and digestion of the trypsin-specific synthetic substrate Chromozym-TRY were extremely alkaline (pH 10). Direct measurements were made of the pH within different compartments of the tsetse digestive tract. We conclude that the gut pH of teneral flies, from the proventriculus to the hindgut, is alkaline, in contradiction to previous measurements indicating that it was mildly acidic. When tsetse flies were analysed 48 h after their first bloodmeal, a pH gradient from the proventriculus (pH 10.6+/-0.6) to the posterior midgut (pH 7.9+/-0.4) was observed.

Understanding reproductive and developmental processes of socioeconomically important parasitic nematodes is of fundamental scientific interest and could have important implications for developing novel methods for parasite control via the disruption or interruption of such processes. Central to investigating reproductive molecular biology is the identification and characterisation of genes with sex-specific expression profiles. However, there is currently a paucity of information on such genes and their expression patterns in parasitic nematodes. This article describes recent progress on the characterisation of sex-specific genes from a parasitic nematode of veterinary importance, and discusses the fundamental scientific and applied implications of this work.

This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively).

p-amidinophenylmethylphosphinic acid (AMPA) was designed, synthesized and crystallized in complex with trypsin to study interactions with the oxyanion hole at the S1 site. In comparison to benzamidine, AMPA shows improved activity, which the crystal structure demonstrates to result from hydrogen bonds between the negatively charged phosphinic acid group and the catalytic residues at the oxyanion hole.

Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.

The crystal structure of a new inhibitor of human neutrophil elastase (HNE), N-[2-[5-(tert-butyl)-1,3,4-oxadiazol-2-yl]-(IRS)-1-(methylethyl)-2-oxoethyl]-2-(5-amino-6-oxo-2-phenyl-6H-pyrimidin-1-ly)acetamide (ONO-6818, 1) complexed to porcine pancreatic elastase (PPE) has been determined at 1.86 A resolution. Analytical results provided evidence of a 1:1 complex in which the electrophilic ketone of 1 covalently bound to O gamma of Ser195 at the active site of PPE. The role of the unique electron-withdrawing ketone of 1 has been elucidated.

A series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds has been synthesized and the inhibitory profile of these compounds toward human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3) was then determined. Most of the compounds were found to be potent, time-dependent inhibitors of elastase, with some of the compounds exhibiting k(inact)/K1 values as high as 4,928,300 M(-1) s(-1). The inhibitory potency of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide platform was found to be influenced by both the pKa and the inherent structure of the leaving group. Proper selection of the primary specificity group (R(I)) was found to lead to selective inhibition of HLE over Cat G, however, those compounds that inhibited HLE also inhibited PR 3, albeit less efficiently. The predictable mode of binding of these compounds suggests that, among closely-related serine proteases, highly selective inhibitors of a particular serine protease can be fashioned by exploiting subtle differences in their S' subsites. This study has also demonstrated that the degradative action of elastase on elastin can be abrogated in the presence of inhibitor 17.

The aim of the study was to evaluate the effect of the protein Seminal Vesicle Protein No. 4 (SV-IV), a potent inhibitor of antithrombin III (antithrombin), on the coagulation of blood obtained from patients affected by hemophilia A. In the coagulating blood of these patients, the antithrombin/thrombin ratio was found to be markedly higher (about 44) than in normal individuals (about 4. 4). This high ratio was related to the low efficiency of thrombin-generating reactions induced by the factor VIII deficiency and to the high levels of free (not bound to serine proteases) antithrombin present in the hemophilic serum (antithrombin concentration was the same in normal and hemophilic plasma). The elevated concentration of free antithrombin in hemophiliacs was primarily a consequence of a reduced consumption caused by the scarce availability in the hemophilic serum of factors Xa and IIa, which are serine proteases possessing strong binding affinity for antithrombin. Addition of SV-IV to coagulating hemophilic blood reduced markedly the serum antithrombin and thrombin-antithrombin complexes, normalizing, as a consequence, the clotting time and other coagulation parameters. Similar results were obtained by using appropriate concentration of factor VIII.

A series of chemically modified mutants (CMMs) of subtilisin B. lentus (SBL) were generated employing the combination of site-directed mutagenesis and chemical modification. This strategy entails the mutation of a selected active site residue to cysteine and its subsequent modification with a methanethiosulfonate reagent CH3SO2S-R, where R may be infinitely variable. The present study was undertaken to evaluate the changes in specificity and pH-activity profiles that could be induced by modification of S156C and S166C in the S1 pocket of SBL with a representative range of side chain modifications, namely R=-CH3, -CH2C6H5, -CH2CH2NH3+ and CH2CH2SO3 . The side chain of S156C is surface exposed and well solvated while that of S166C points into the pocket. Kinetic evaluation of the CMMs with suc-AAPF-pNA as substrate showed that the kcat/K(M)s changed very little for the S156C CMMs, but varied by up to 11-fold for the S166C CMMs. pH-Activity profiles were also determined, and showed that a negatively or positively charged side chain modification increased or decreased respectively, the pKa of the catalytic triad histidine for both modification sites but with more dramatic changes for the interior pointing S166C than for the solvent exposed S156C site. As an additional probe of altered specificity, inhibition of the CMMs by a representative series of 5 boronic acid transition state analogue inhibitors was determined. The K(I)s observed ranged from a 3.5-fold improvement over the WT value, to a 12-fold decrease in binding. Overall, greater variability in all the parameters measured, activity, pKa, and boronic acid binding resulted from modification at the inward pointing 166 site than at the solvent-exposed 156 site.

Arthropod hemolymph contains proteins with serine proteinase inhibitory activity. These inhibitors may exist in plasma or in hemocyte granules. Serine proteinase inhibitors from the Kazal, Kunitz, alpha-macroglobulin, and serpin families have been identified in arthropod hemolymph and have been characterized biochemically. Two new families of low molecular weight serine proteinase inhibitors have recently been discovered: one in silkworms (the Bombyx family) and another in locusts and a crayfish. The serine proteinase inhibitors in arthropod hemolymph are likely to function in protecting their hosts from infection by pathogens or parasites. Some may inhibit fungal or bacterial proteinases. Others probably have roles in regulating endogenous proteinases involved in coagulation, prophenol oxidase activation, or cytokine activation.

The amidine-containing alpha-aminoalkyl phosphonofluoridate 3 (Cbz-(4-AmPhGly)P(OPh)(F)) is a very potent inhibitor of trypsin-like enzymes. It was prepared by hydrolyzing the corresponding phosphonate diphenyl ester 4 followed by reaction of fluoride with the phosphonochloridate prepared from the intermediate phosphonic acid monoester 5. Compound 3 is the most potent amidine-containing organophosphorus inhibitor yet reported for trypsin-like enzymes. It inhibits trypsin and thrombin with second-order rate constants (Kobs/[I]) of 2.6 x 10(5) M-1 s-1 and 1.0 x 10(5) M-1 s-1, respectively, showing a 130-fold and a 1250-fold rate enhancement over the corresponding diphenyl ester (4). It also inactivates trypsin 2 orders of magnitude more potently than simple phosphonofluoridates such as DFP,1 Sarin and Soman. The phosphonofluoridate 3 does not inhibit other serine proteases such as porcine pancreatic elastase (PPE) and the esterase acetylcholinesterase (AChE). The phosphonofluoridate 3 is hydrolyzed rapidly in buffer solution and has a t1/2 of 4.5 s at pH 7.5.

A series of new trypsin-like serine protease inhibitors, 1, 2 and 7-23, containing amidinobenzene moiety was found to show potent LTB4-receptor affinity. Among them, compounds 1 and 2 were found to be LTB4 receptor antagonists based on an inhibition assay of human polymorphonuclear neutrophil (PMN) intracellular calcium mobilization induced by LTB4. Compounds 1 and 2, which satisfy the reported structural requirements for good oral activity, are expected to show a balanced dual mode of action, i.e., protease inhibitory activity and LTB4 receptor antagonist activity, in vivo.

A novel series of pivaloyloxy benzene derivatives has been identified as potent and selective human neutrophil elastase (HNE) inhibitors. Convergent syntheses were developed in order to identify the inhibitors which are intravenously effective in an animal model. A compound of particular interest is the sulfonanilide-containing analogues. Structure-activity relationships are discussed. Structural requirements for metabolic stabilization are also discussed.

The aim of this paper was to investigate the influence of preparation parameters on gelatin microspheres production, chemico-physical characteristics and drug encapsulation. In particular, we focussed our attention on the manufacturing parameters such as amount of polymer, stirring speed, presence and concentration of surfactants. As model drugs, TAPP-Br, clonidine hydrochloride and bromocriptine mesylate were chosen in order to compare their encapsulation and release characteristics with microspheres. In the second part of this work, a study of the influence of thermal treatment on the microspheres is reported, performed with the aim to possibly modify gelatin dissolution and drug release. In particular, the effect of this treatment was evaluated on microsphere characteristics such as swelling, porosity and dissolution, and finally on the release profiles of the encapsulated drugs.

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P1 residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P1 residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P1 alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.

In an attempt to explore the process of naturally occurring secretory leukoprotease inhibitor (SLPI) fragmentation, the cleavage profile of SLPI, which had been prepared by recombinant techniques, was investigated biochemically. Restricted fragments of SLPI were detected using SDS-PAGE after treatment with human neutrophil elastase (NE) or normal saliva and sequenced at their cleavage sites. Among these restricted fragments, two species of nearly half-length SLPIs that contained the C-terminal domain, (Arg58-Ala107)SLPI and (Arg59-Ala107)SLPI, were detected. They were both as active at inhibiting NE as the parent SLPI. These results suggest that functional SLPI derivatives may be generated physiologically in the respiratory tract under inflammatory and healthy conditions.

The beta-amyloid precursor protein is the precursor of the main component of senile plaques (the beta-amyloid peptide or beta/A4) found in the brain of aged humans and, in higher amounts, in the brain of Alzheimer's disease and Down's syndrome subjects. Four different forms of beta-amyloid precursor protein messenger RNAs have been described in humans and rodents: beta-amyloid precursor protein 695, beta-amyloid precursor protein 714, beta-amyloid precursor protein 751 and beta-amyloid precursor protein 770 messenger RNAs (numbers corresponding to the number of encoded amino acids). The two latter forms are characterized by containing in their sequence a region with high homology to the Kunitz family of serine protease inhibitors. We have used oligonucleotide probes to study the distribution of the different messenger RNAs encoding each of the four beta-amyloid precursor proteins by in situ hybridization histochemistry in human, rat and mouse brain. We found that beta-amyloid precursor protein 695, beta-amyloid precursor protein 714 and beta-amyloid precursor protein 751 messenger RNAs were widely distributed in the human, rat and mouse brain and that their distribution was roughly similar in most brain areas in these three species. The distribution of beta-amyloid precursor protein 770 messenger RNA was not so wide and differed among the three species studied. beta-amyloid precursor protein 751 and 770 messenger RNAs were the only forms present at significant levels in rodent choroid plexus and meninges, while beta-amyloid precursor protein messenger RNA isoforms containing and lacking the Kunitz domain were detected in the human choroid plexus. We also observed that the relative levels of beta-amyloid precursor protein 751 and 770 messenger RNAs in the rat cerebral white matter as well as in the mouse and human striatum were higher than those of the beta-amyloid precursor protein messenger RNAs lacking the Kunitz domain. While the most abundant beta-amyloid precursor protein messenger RNAs in the brain of all three species under study were, in descending order, beta-amyloid precursor protein 695 and beta-amyloid precursor protein 751 messenger RNAs, the least abundant form was not the same for all species: in human it was beta-amyloid precursor protein 714 messenger RNA and in rat and mouse brain it was beta-amyloid precursor protein 770 messenger RNA. Our results show differences both inter- and intraspecies of the relative abundance and distribution of four beta-amyloid precursor protein messenger RNAs in rat, mouse and human brain.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G, and pancreatic elastase by the hemolymph of 14 insect species in six orders has been investigated. 2. All samples showed great diversity in terms of both total proteinase inhibitory capacity and specificity. 3. The highest total inhibitory capacity was found in the larval hemolymph of species in the beetle family Tenebrionidae and the lowest in that of an adult coreid bug, Acanthocephala femorata.

The recent finding that the serine protease inhibitor, alpha 1-antichymotrypsin, is tightly associated with the amyloid deposits in brains of normal aged individuals and patients with Alzheimer's disease [Abraham C. R., Selkoe D. J. and Potter H. (1988) Cell 52, 487-501], suggests a role for this inhibitor in the progressive deposition of brain amyloid in humans. We have used immunocytochemistry to detect alpha 1-antichymotrypsin in the amyloid that accumulates in brains of aged monkeys, a naturally occurring animal model of Alzheimer-like neuropathology. In monkeys of increasing age, the earliest alpha 1-antichymotrypsin immunoreactivity was found in cortical perivascular cells, before the appearance of either Thioflavin S-detectable amyloid deposits or beta-protein reactivity in the vessel walls. Subsequently, amyloid deposits appeared in small meningeal blood vessels and cortical neuritic plaques. The oldest monkeys also showed microvascular amyloid in the cortical gray matter. Amyloid was never seen in white matter. The amyloid deposits in meningeal vessels were always positive for both beta-protein and alpha 1-antichymotrypsin, whereas in the cortex, alpha 1-antichymotrypsin immunoreactivity seemed to appear somewhat later than that of beta-protein. These findings demonstrate that two of the brain amyloid components of human senescence and Alzheimer's disease--the beta-protein and the protease inhibitor alpha 1-antichymotrypsin--are also present in the amyloid deposits of normal aged monkey brain. The extended molecular parallels between normal brain aging and Alzheimer's disease suggest that similar biochemical mechanisms may underlie progressive amyloid deposition in both situations.