Alpha-1 antichymotrypsin is involved in astrocyte injury in concert with arginine-vasopressin during the development of acute hepatic encephalopathy.

BACKGROUND AND AIMS: We developed a bio-artificial liver (BAL) using a radial-flow bioreactor and rescued mini-pig models with lethal acute liver failure (ALF). The point of the rescue is the recovery from hepatic encephalopathy (HE). HE on ALF has sometimes resulted in brain death following brain edema with astrocyte swelling. Several factors, including ammonia and glutamine, have been reported to induce astrocyte swelling and injury. However, many clinicians believe that there are any other factors involved in the development of HE. Therefore, the aim of this study was to identify novel HE-inducible factors, particularly those inducing astrocyte dysfunction. METHODS: Mini-pig plasma samples were collected at three time points: before the administration of toxins (α-amanitin and LPS), when HE occurred after the administration of toxins, and after treatment with extracorporeal circulation (EC) by the BAL. To identify the causative factors of HE, each plasma sample was subjected to a comparative proteome analysis with two-dimensional gel electrophoresis and mass spectrometry. To assess the direct effects of candidate factors on the astrocyte function and injury, in vitro experiments with human astrocytes were performed. RESULTS: Using a proteome analysis, we identified alpha-1 antichymotrypsin (ACT), which was increased in plasma samples from mini-pigs with HE and decreased in those after treatment with EC by BAL. In in vitro experiments with human astrocytes, ACT showed growth-inhibitory and cytotoxic effects on astrocytes. In addition, the expression of water channel protein aquaporin-4, which is induced in injured astrocytes, was increased following ACT treatment. Interestingly, these effects of ACT were additively enhanced by adding arginine-vasopressin (AVP) and were canceled by adding an AVP receptor antagonist. CONCLUSIONS: These results suggest that ACT is involved in astrocyte injury and dysfunction in concert with AVP during the development of acute HE.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

α1-Antichymotrypsin inactivates staphylococcal cysteine protease in cross-class inhibition.

Staphylococcal cysteine proteases are implicated as virulence factors in human and avian infections. Human strains of Staphylococcus aureus secrete two cysteine proteases (staphopains A and B), whereas avian strains express staphopain C (ScpA2), which is distinct from both human homologues. Here, we describe probable reasons why the horizontal transfer of a plasmid encoding staphopain C between avian and human strains has never been observed. The human plasma serine protease inhibitor α(1)-antichymotrypsin (ACHT) inhibits ScpA2. Together with the lack of ScpA2 inhibition by chicken plasma, these data may explain the exclusively avian occurrence of ScpA2. We also clarify the mechanistic details of this unusual cross-class inhibition. Analysis of mutated ACHT variants revealed that the cleavage of the Leu383-Ser384 peptide bond results in ScpA2 inhibition, whereas hydrolysis of the preceding peptide bond leads to ACHT inactivation. This evidence is consistent with the suicide-substrate-like mechanism of inhibition.

Alpha1-antichymotrypsin induces TNF-alpha production and NF-kappaB activation in the murine N9 microglial cell line.

Microglia are known to accumulate in senile plaques of Alzheimer's disease (AD) together with a set of proteins including alpha(1)-antichymotrypsin (ACT). To investigate the biological effects of the interaction between ACT and microglia, we examined cytokine production by the murine N9 microglial cell line after ACT treatment. Real-time PCR analysis and specific immunoassays demonstrate that ACT triggers mRNA expression and release of TNF-alpha by N9 microglial cells. Furthermore, we show that ACT induces a significant increase in NF-kappaB nuclear translocation. Taken together, these data demonstrate that ACT might contribute to the inflammatory mechanisms present in AD senile plaques.

The inflammation-sensitive protein alpha 1-anti-chymotrypsin neutralizes fibrillar aggregation and cytotoxicity of the beta-amyloid peptide more effectively than alpha 1-antitrypsin.

OBJECTIVES: A neuroinflammatory process, triggered by amyloid-beta (Abeta)-peptide, is thought to play a central role in the neurodegenerative process leading to Alzheimer's disease (AD). Abeta(25-35) retains the functionality of Abeta(42) and was employed to investigate the effects of inflammation-sensitive proteins (ISPs) alpha1-antichymotrypsin (A1ACT) and alpha1-antitrypsin (A1AT) on fibrillar aggregation and cytotoxicity. DESIGN AND METHODS: Inhibitory concentrations of the ISPs were determined in an established human red blood cell lysis model of Abeta-cytotoxicity. For studies of Abeta-fibrillar aggregation CSF levels of A1ACT (0.041 microM)/A1AT (0.11 microM) were incubated with Congo Red dye 25 microM+Abeta(25-35) 10 microM noting the formation of visible aggregates and spectrophotometric changes over 24 h. RESULTS: A1ACT at CSF reported levels inhibited fibrillar aggregation and cytotoxicity while A1AT at CSF reported levels failed to cause a similar inhibition. CONCLUSIONS: A1ACT neutralizes fibrillar aggregation and cytotoxicity of Abeta-peptide more effectively than A1AT. Both proteins are known to be co-deposited with Abeta within senile plaques of AD brains.

Prostate-specific antigen stimulates osteoprotegerin production and inhibits receptor activator of nuclear factor-kappaB ligand expression by human osteoblasts.

BACKGROUND: Prostate cancer cells produce a large amount of prostate-specific antigen (PSA), which is widely used as a marker for this cancer. Even though it is widely used in the diagnosis of prostate cancer, many aspects of the pathophysiologic role of PSA in bone metastasis remain obscure. The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the activation of osteoclasts, while osteoprotegerin (OPG) neutralizes the action of RANKL. Various substances that act on bone have been shown to modulate the production of RANKL and OPG by osteoblasts. METHODS: In this study, we investigated the effect of PSA on the expression of OPG and RANKL mRNA and on protein production in human osteoblast-like cells. RESULTS: After addition of PSA and culture for 72 h, OPG mRNA expression and protein secretion by MG-63 and SaOS-2 cells showed a concentration-dependent increase. When osteoblasts were incubated with PSA (100 ng/ml), OPG mRNA expression and protein secretion increased with the passage of time. alpha1 -antichymotrypsin (ACT), which inactivates the serine protease activity of PSA, inhibited the increase of OPG mRNA expression and protein production in response to PSA, and this effect of PSA was also inhibited by anti-transforming growth factor-beta antibody. CONCLUSIONS: Based on our findings, PSA acts on human osteoblast-like cells via its own serine protease activity and promotes osteoblast differentiation. In addition, PSA stimulates OPG production and inhibits RANKL expression of osteoblasts, and inhibits bone resorption by osteoclasts, suggesting that it contributes to the characteristic osteoblastic features of bone metastases of prostate cancer.

Alpha1-antichymotrypsin, an inflammatory protein overexpressed in Alzheimer's disease brain, induces tau phosphorylation in neurons.

Amyloid plaques and neurofibrillary tangles are key pathological features of Alzheimer's disease. Alzheimer's disease pathology is also characterized by neuroinflammation and neuronal degeneration, with the proteins associated with inflammatory responses being found in tight association with the plaques. One such protein is the serine protease inhibitor alpha-1-antichymotrypsin (ACT). ACT has been shown to promote Abeta polymerization in vitro and in vivo, and levels of ACT protein in plasma and cerebrospinal fluid from Alzheimer's patients have been found to correlate with progression of dementia. Here we investigated the possible involvement of ACT in tau phosphorylation and tangle formation. As was previously found for Alzheimer's disease, brains from patients with non-Alzheimer's tauopathies exhibited an enhanced expression of ACT, which correlated with the level of tau hyperphosphorylation. Transgenic mice expressing human ACT alone or ACT along with mutant human amyloid precursor protein (APP) showed a significant increase in tau phosphorylation, suggesting that this inflammatory protein can induce tau hyperphosphorylation. The increase in phosphorylation was observed at PHF-1 (P-Ser396/P-Thr404), P-Ser202 and P-Thr231 sites on tau, the P-tau epitopes that are associated with tangles in the patients. This result was further confirmed by the finding that addition of purified ACT induced the same Alzheimer's disease-related tau hyperphosphorylation in cortical neurons cultured in vitro. This correlated with an increase in extracellular signal regulated kinase (ERK) and glycogen synthase kinase-3 activation, indicating their involvement in ACT-induced tau phosphorylation. The ACT-treated neurons showed neurite loss and subsequently underwent apoptosis. Approximately 40-50% of neurons were TUNEL positive by 6 and at 24 h >70% of the neurons showed staining suggesting that ACT was inducing apoptosis in these neurons. These findings indicate that inappropriate inflammatory responses are a potential threat to the brain and that intervention directed at inhibiting the expression or function of ACT could be of therapeutic value in neurodegenerative diseases such as Alzheimer's and other tauopathies.

Astrocyte-specific expression of the alpha1-antichymotrypsin and glial fibrillary acidic protein genes requires activator protein-1.

An amyloid-associated serine proteinase inhibitor (serpin), alpha(1)-antichymotrypsin (ACT), is encoded by a gene located within the distal serpin subcluster on human chromosome 14q32.1. The expression of these distal serpin genes is determined by tissue-specific chromatin structures that allow their ubiquitous expression in hepatocytes; however, their expression is limited to a single ACT gene in astrocytes. In astrocytes and glioma cells, six specific DNase I-hypersensitive sites (DHSs) were found located exclusively in the 5'-flanking region of the ACT gene. We identified two enhancers that mapped to the two DHSs at -13 kb and -11.5 kb which contain activator protein-1 (AP-1) binding sites, both of which are critical for basal astrocyte-specific expression of ACT reporters. In vivo, these elements are occupied by c-jun homodimers in unstimulated cells and c-jun/c-fos heterodimers in interleukin-1-treated cells. Moreover, functional c-jun is required for the expression of ACT in glioma cells because both transient and stable inducible overexpression of dominant-negative c-jun(TAM67) specifically abrogates basal and reduces cytokine-induced expression of ACT. Expression-associated methylation of lysine 4 of histone H3 was also lost in these cells, but the DHS distribution pattern and global histone acetylation were not changed upstream of the ACT locus. Interestingly, functional AP-1 is also indispensable for the expression of glial fibrillary acidic protein (GFAP), which is an astrocyte-specific marker. We propose that AP-1 is a key transcription factor that, in part, controls astrocyte-specific expression of genes including the ACT and GFAP genes.

The serpins alpha-1-antitrypsin and alpha-1-antichymotrypsin specifically interact with immunophilins.

In a yeast two-hybrid screen FKBP13, a member of the FK506 Binding Protein (FKBP) family, was detected to interact with the serpin alpha-1-antichymotrypsin (ACT). The specificity of the interaction was confirmed in vitro and by the lack of interaction of ACT with FKBP25 and FKBP52. Mutational analysis of ACT revealed that the entire protein is necessary to interact with FKBP13. ACT but also different unrelated small regions of the ACT protein were able to interact with the smaller FKBP12, demonstrating a rather nonspecific interaction with this immunophilin. Naturally occuring mutants of ACT were able to interact as well. Antitrypsin (AT) closely related to ACT did only interfere with FKBP12 a protein that does presumably not reside in the same cellular compartment with AT and ACT. Both serpins interacted with the unrelated immunophilin cyclophilin A. In conclusion the serpin alpha-1-antichymotrypsin physiologically interacts with the ER-immunophilin FKBP13 and the secreted immunophilin cyclophilin A in vivo whereas alpha-1-antitrypsin might only react with cyclophilin A; both serpins may be controlled thereby in their genuine function.

Glioma cell activation by Alzheimer's peptide Abeta1-42, alpha1-antichymotrypsin, and their mixture.

We compared the effects ofAlzheimer's peptide (Abeta1-42), a,-antichymotrypsin (ACT) and an ACT/Abeta1-42 mixture on human glioma DK-MG cells. The solution of Abeta (5 microM) formed by 2-h incubation at room temperature induced tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 levels by 55 and 45%, respectively, and increased gelatinase B activity by 67%, while exposure of cells to the ACT/Abeta1-42 mixture (1:10 molar ratio ACT: Abeta1-42) under the same experimental conditions showed no effect on IL-6 levels or gelatinase B activity, but strongly induced TNF-alpha (by 190%), compared to the controls. Stimulation of the cells with Abeta1-42 alone, but not with ACT, increased by about 20% low-density lipoprotein (LDL) uptake and mRNA levels for LDL receptor and HMG-CoA reductase, while the ACT/Abeta1-42 mixture significantly increased LDL uptake (by 50%), up-regulated mRNA levels for LDL receptor and HMG-CoA reductase by 48 and 63%, respectively, and increased lipid accumulation by about 20-fold. These data suggest a possible new role for Abeta in Alzheimer's disease through its interaction with the inflammatory reactant, ACT.

Inhibition of prostate-specific antigen (PSA) by alpha(1)-antichymotrypsin: salt-dependent activation mediated by a conformational change.

Prostate-specific antigen (PSA) and its SDS-stable complex with the serine proteinase inhibitor (serpin) alpha(1)-antichymotrypsin (ACT), which is the dominant form of PSA in serum, are in widespread use as markers for the diagnosis of prostate cancer, and there is increasing evidence for the involvement of PSA proteinase activity itself in the development of prostate and other cancers. However, both the formation and degradation of the PSA-ACT complex, denoted PSA*ACT* to indicate substantial changes in the structure of both proteins on complex formation, have been incompletely studied. Here we determine rate and equilibrium constants for the steps involved in PSA*ACT* formation and demonstrate that (a) the effects of added NaCl, polyamines, and Zn(2+) on this process parallel their effects on PSA catalytic activity [Hsieh, M.-C., and Cooperman, B. S. (2000) Biochim. Biophys. Acta 1481, 75-87], (b) the effect of added NaCl in dramatically increasing the rate of ACT inhibition of PSA correlates with salt-induced changes in PSA conformation, and (c) the PSA*ACT* complex is subject to proteolysis by human neutrophil elastase. Possible clinical implications of these findings are considered.

Alpha1-antichymotrypsin/Alzheimer's peptide Abeta(1-42) complex perturbs lipid metabolism and activates transcription factors PPARgamma and NFkappaB in human neuroblastoma (Kelly) cells.

Amyloid-beta peptide (Abeta) and the serpin proteinase inhibitor alpha1-antichymotrypsin (ACT) are components of the amyloid plaques associated with Alzheimer's disease (AD). Abeta exists in soluble monomeric and oligomeric forms and in an insoluble polymerised fibrillar form, but it is not clear which of these plays the most important role in the etiology of AD. In vitro, Abeta(1-42) interacts with ACT, and as a result of this, ACT loses its proteinase inhibitor activity and polymerisation of Abeta(1-42) is promoted. Here we provide evidence that new molecular forms resulting from incubation of ACT with Abeta(1-42) have multiple cellular level effects on neuronal cells. The mixture of soluble Abeta and an ACT/Abeta complex formed by 2 hr incubation at a 10:1 molar ratio of Abeta:ACT strongly induce cellular proliferation and expression of transcription factors peroxisome proliferator-activated receptor-gamma (PPARgamma) and NFkappaB, and also increase uptake and depress degradation of native and oxidised low-density lipoprotein (LDL) by cells. Similar but less pronounced effects are seen when cells are exposed to the Abeta peptide alone preincubated for 2 hr. Abeta(1-42) and to a lesser extent ACT/Abeta(1-42) complex mixture prepared by 2 hr incubation both inhibit association of native LDL with cells. Neither ACT alone nor the Abeta(1-42) and ACT/Abeta(1-42) forms prepared by 24-hr incubation show any significant effects in these assays. We propose that specific molecular forms of Abeta(1-42) and ACT/Abeta(1-42) complex mixture, both dependent on the abundances of Abeta(1-42) and ACT/Abeta(1-42) in vivo and on their time of exposure to each other, have cellular effects which are important for the initiation and progression of the pathologies associated with AD.

Release of soluble tryptase but only minor amounts of chymase activity from cutaneous mast cells.

Tryptase and chymase are the major serine proteinases of skin mast cells but their biologic significance depends on their activity. In this study, we demonstrate the release of soluble activity of tryptase, but not markedly that of chymase, into skin blister fluids induced by freezing with liquid nitrogen as well as into supernatant during incubation of 8 whole skin specimens with compound 48/80 for up to 2 days followed by sonication. Incubation of 3 other skin specimens in compound 48/80 for up to 2 days revealed that the number of mast cells displaying tryptase activity decreased significantly on day 2, and the number of mast cells showing chymase activity (but not those showing chymase immunoreactivity) decreased significantly on day 1 but not thereafter on day 2. The results of 3 skin organ cultures for up to 14 days showed steady decrease in the number of tryptase-positive cells but persistence of mast cells containing chymase activity. Chymase in solution was sensitively inhibited by 0.01 mg/ml alpha1-antichymotrypsin but higher concentrations (0.3-3.0 mg/ml) were needed for inhibiting chymase on skin sections. In conclusion, after mast cell degranulation tryptase activity is substantially solubilized and it may potentially affect both local and distant skin structures. Instead, chymase is partially inactivated and the remaining chymase activity persists at the site of degranulation having only local effects.

A novel approach for studying endogenous abeta processing using cultured primary neurons isolated from APP transgenic mice.

The central component of senile amyloid plaques in Alzheimer's disease (AD) is the beta-amyloid peptide (Abeta), derived from proteolytic processing of the amyloid precursor protein (APP). In this study, we developed an in vitro model to measure and identify soluble Abeta from primary cortical neurons. Neurons were isolated from mice transgenic for human APP695 containing the K670N, M671L double mutation. We characterized soluble Abeta using Western blot and ELISA assays. We found that the Abeta levels in conditioned media from these neurons were readily detectable and almost five times higher than in CSF. The majority of Abeta in the media was Abeta1-40; however, Abeta1-42 was also detectable. When the neurons were exposed to Phorbol 12-myristate 13-acetate (PMA), alpha1-antichymotrypsin, or alpha1-antitrypsin, the alterations of soluble Abeta levels were consistent with other models reported. Most importantly, the soluble Abeta in our model was remarkably stable, and aliquots were unchanged after prolonged incubations or repeated freeze/thaw cycles. The Abeta appeared to be monomeric by Western blot analysis. Soluble Abeta coimmunoprecipitated with endogenous mouse apolipoprotein E from the primary cultures. Taken together, our data demonstrated that using a Western blot assay to detect soluble Abeta from transgenic mouse overexpressing APP695 is sensitive, specific, and reliable and provides an accessible model for examining the neuronal metabolism of APP and Abeta.

alpha 1-Antichymotrypsin is the human plasma inhibitor of macrophage ectoenzymes that cleave pro-macrophage stimulating protein.

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.

Alpha-1-antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease.

Alpha(1)-antichymotrypsin (ACT), an acute-phase inflammatory protein, is an integral component of the amyloid deposits in Alzheimer's disease (AD) and has been shown to catalyze amyloid beta-peptide polymerization in vitro. We have investigated the impact of ACT on amyloid deposition in vivo by generating transgenic GFAP-ACT-expressing mice and crossing them with the PDGF-hAPP/V717F mice, which deposit amyloid in an age-dependent manner. The number of amyloid deposits measured by Congo Red birefringence was increased in the double ACT/amyloid precursor protein (APP) transgenic mice compared with transgenic mice that only expressed APP, particularly in the hippocampus where ACT expression was highest, and the increase was preceded by elevated total amyloid beta-peptide levels at an early age. Our data demonstrate that ACT promotes amyloid deposition and provide a specific mechanism by which inflammation and the subsequent upregulation of astrocytic ACT expression in AD brain contributes to AD pathogenesis.

Astroglial expression of human alpha(1)-antichymotrypsin enhances alzheimer-like pathology in amyloid protein precursor transgenic mice.

Proteases and their inhibitors play key roles in physiological and pathological processes. Cerebral amyloid plaques are a pathological hallmark of Alzheimer's disease (AD). They contain amyloid-ss (Ass) peptides in tight association with the serine protease inhibitor alpha(1)-antichymotrypsin.(1,2) However, it is unknown whether the increased expression of alpha(1)-antichymotrypsin found in AD brains counteracts or contributes to the disease. We used regulatory sequences of the glial fibrillary acidic protein gene(3) to express human alpha(1)-antichymotrypsin (hACT) in astrocytes of transgenic mice. These mice were crossed with transgenic mice that produce human amyloid protein precursors (hAPP) and Ass in neurons.(4,5) No amyloid plaques were found in transgenic mice expressing hACT alone, whereas hAPP transgenic mice and hAPP/hACT doubly transgenic mice developed typical AD-like amyloid plaques in the hippocampus and neocortex around 6 to 8 months of age. Co-expression of hAPP and hACT significantly increased the plaque burden at 7 to 8, 14, and 20 months. Both hAPP and hAPP/hACT mice showed significant decreases in synaptophysin-immunoreactive presynaptic terminals in the dentate gyrus, compared with nontransgenic littermates. Our results demonstrate that hACT acts as an amyloidogenic co-factor in vivo and suggest that the role of hACT in AD is pathogenic.

DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor.

This paper explores the possibility that neutrophil-derived DNA interferes with the inhibition of neutrophil cathepsin G (cat G) and proteinase 3 by the lung antiproteinases alpha(1)-proteinase inhibitor (alpha(1)PI), alpha(1)-antichymotrypsin (ACT), and mucus proteinase inhibitor (MPI). A 30-base pair DNA fragment ((30bp)DNA), used as a model of DNA, tightly binds cat G (K(d), 8.5 nM) but does not react with proteinase 3, alpha(1)PI, ACT, and MPI at physiological ionic strength. The polynucleotide is a partial noncompetitive inhibitor of cat G whose K(i) is close to K(d). ACT and alpha(1)PI are slow binding inhibitors of the cat G-(30bp)DNA complex whose second-order rate constants of inhibition are 2300 M(-1) s(-1) and 21 M(-1) s(-1), respectively, which represents a 195-fold and a 3190-fold rate deceleration. DNA thus renders cat G virtually resistant to inhibition by these irreversible serpins. On the other hand, (30bp)DNA has little or no effect on the reversible inhibition of cat G by MPI or chymostatin or on the irreversible inhibition of cat G by carbobenzoxy-Gly-Leu-Phe-chloromethylketone. The polynucleotide neither inhibits proteinase 3 nor affects its rate of inhibition by alpha(1)PI. These findings suggest that cat G may cause lung tissue destruction despite the presence of antiproteinases.

Alpha 1-antichymotrypsin inhibits A beta degradation in vitro and in vivo.

The neuropathology of Alzheimer's disease (AD) is characterized by extensive deposition of the toxic amyloid beta peptide (A beta) in selected regions of the brain and brain vasculature (Selkoe, 1999). Thus, lowering the levels of A beta may be beneficial for AD patients. A beta is a proteolytic fragment derived from the amyloid precursor protein (APP). The mechanisms of A beta formation from its precursor have been studied extensively; however, considerably less effort has been invested into studying A beta clearance. We find that the degradation of A beta in our system is dependent upon the presence of a metallopeptidase E.C.3.4.24.15 (MP24.15) (Yamin et al., 1999). We have previously purified MP24.15 to homogeneity from AD brain and identified it as an APP-processing protease in vitro (Papastoitsis, 1994). To confirm its role in cell culture, we transfected SKNMC neuroblastoma cells with sense and antisense cDNAs of MP24.15 and with a mock construct. Compared to mock conditioned media (CM), CM of MP24.15-overexpressing cells had very high A beta-degrading activity. Conversely, CM of antisense-expressing cells lacked A beta-degrading activity. These results suggested that MP24.15 is involved in A beta degradation. Characterization of the proteolytic activity directly responsible for A beta degradation using a spectrum of protease inhibitors revealed that only serine protease inhibitors completely blocked A beta degradation. Therefore, MP24.15 appears to activate a serine protease, which then cleaves A beta. Interestingly, alpha 1-antichymotrypsin (ACT) which we discovered to be highly elevated in AD brain (Abraham, et al., 1988) also inhibited A beta degradation. To our delight, ACT proved to be an inhibitor of A beta degradation in vivo as well. When we crossed transgenic mice expressing human ACT with plaque-producing mice expressing human APP, the doubly transgenic mice had twice as many plaques at 20 months of age as the APP mice (Mucke et al., 2000). Successful completion of this study could lead to the design of reagents that would reduce the amyloid load in AD patients.

Glial-derived proteins activate cultured astrocytes and enhance beta amyloid-induced glial activation.

A prominent feature of Alzheimer's disease (AD) pathology is an abundance of activated glia (astrocytes and microglia) in close proximity to the amyloid plaques. These activated glia overexpress a number of proteins that may participate in the progression of the disease, possibly by propagation of inflammatory and oxidative stress responses. The beta-amyloid peptide 1-42 (Abeta), a major constituent of neuritic plaques, can itself induce glial activation. However, little is known about whether other plaque components, especially the upregulated glial proteins, can induce glial activation or modulate the effects of Abeta on glia. In this study, we focused on four glial proteins that are abundant in amyloid plaques and/or that are known to interact with Abeta: alpha1-antichymotrypsin (ACT), interleukin-1beta (IL-1beta), S100beta, and butyrylcholinesterase (BChE). We examined the ability of these proteins to activate rat cortical astrocyte cultures and to influence the ability of Abeta to activate astrocytes. Treatment of astrocytes with ACT, IL-1beta, or S100beta resulted in glial activation, as assessed by reactive morphology, upregulation of IL-1beta, and production of inducible nitric oxide synthase and nitric oxide. The ability of Abeta to induce astrocyte activation was also enhanced in the presence of each of these three proteins. In contrast, BChE alone did not activate astrocytes and had no effect on Abeta-induced activation. These results suggest that certain proteins produced by activated glia may contribute to the chronic glial activation seen in AD through their ability to stimulate astrocytes directly or through their ability to modulate Abeta-induced activation.