Bioengineered xylanase from Misiones Argentina rainforest: A bakery enhancement approach.

In this study, we pursued the heterologous expression of the xylanase gene from Trichoderma atroviride, a native fungus in the province of Misiones, and used it to enhance the textural properties of baked goods through varying enzymatic concentrations. This marks the inaugural exploration into its functionality in the context of bread production. The recombinant xylanase exhibited improved activity, reaching 36,292 U L-1, achieved by supplementing the culture medium with dextrose. Following the optimization of recombinant xylanase concentration, promising results emerged, notably reducing hardness and chewiness parameters of bread significantly. Our findings underscore the potential of this native fungal enzyme for industrial processes, offering a sustainable and efficient means to enhance the quality of baked goods with broad implications for the food industry. No prior research has been documented on the heterologous expression of the xylanase gene derived from T. atroviride, from the Misiones rainforest, expressed in Kluyveromyces lactis. PRACTICAL APPLICATION: This research, focusing on the isolation and cloning of xylanase enzyme from Trichoderma atroviride, a native fungus in the province of Misiones, offers a valuable tool for improving the texture of bakery products. By optimizing enzyme concentrations, our findings present a practical approach for the food industry, offering a viable solution to improve the overall quality and consumer satisfaction of bakery products.

C5aR1 signaling promotes region- and age-dependent synaptic pruning in models of Alzheimer's disease.

INTRODUCTION: Synaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD. METHODS: A combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD. RESULTS: Genetic ablation or pharmacological inhibition of C5aR1 partially rescues excessive pre-synaptic pruning and synaptic loss in an age and region-dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP). DISCUSSION: Reduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD. HIGHLIGHTS: C5aR1 ablation restores long-term potentiation in the Arctic model of AD. C5aR1 ablation rescues region specific excessive pre-synaptic loss. C5aR1 antagonist, PMX205, rescues VGlut1 loss in the Tg2576 model of AD. C1q tagging is not sufficient to induce VGlut1 microglial ingestion. Astrocytes contribute to excessive pre-synaptic loss at late stages of the disease.

C5aR1 signaling promotes region and age dependent synaptic pruning in models of Alzheimer's Disease.

INTRODUCTION: Synaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD. METHODS: A combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD. RESULTS: Genetic ablation or pharmacological inhibition of C5aR1 rescues the excessive pre-synaptic pruning and synaptic loss in an age and region dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP). DISCUSSION: Reduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD.

Purification and characterization of a fungal laccase expressed in Kluyveromyces lactis suitable for baking.

Laccase enzyme can replace chemical additives to improve texture properties and the volume of bread. Laccase encoding gene from Phlebia brevispora, a native fungus from Misiones, Argentina, was expressed in the generally recognized as safe yeast Kluyveromyces lactis. To improve laccase activity, medium conditions were optimized. The use of iron sulfate at a concentration of 1 mM led to optimum laccase activity (1289 U·L-1 ) on the fourth day of incubation. SDS-PAGE analysis revealed that the molecular mass of purified laccase was about 180 kDa. Optimum pH for the enzyme was 4 and optimum temperature was 40°C. Laccase exhibited high stability at low pH and high temperature. The application of recombinant laccase to bread decreased hardness, gumminess, and chewiness and increased bread volume. Based on these results, recombinant laccase from P. brevispora with improved yield is a good option for application as an improver of the physicochemical quality of bread at the industrial level. Besides, it will allow us to advance toward our goal of developing healthy alternatives for the bakery industry. No previous work has been reported concerning the heterologous expression of the laccase gene native to the province of Misiones, Argentina, with an aim for application in baking. PRACTICAL APPLICATION: Healthy bakeries became a trend in recent years. The use of the laccase enzyme increases the specific volume and decreases the hardness of bread, being thus an alternative for the replacement of chemical additives in the bakery industry.

Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression.

BACKGROUND: The complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimer's disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation. METHODS: To elucidate the effects of C5a-C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a-overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7-10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states. RESULTS: ArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a-C5aR1 signaling. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a-C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers. CONCLUSION: C5a-C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD.

Exploring novel Penicillium lipolytic activity from the Paranaense rainforest.

In this study, we evaluated the presence of lipases in twenty fungal strains of the genus Penicillium using an efficient and low-cost method with a view to an application in the treatment of cooking oil residues. The Paranaense rainforest is one of the most biodiverse places on the planet, making it the most likely site to find new fungal strains with lipolytic potential. The objective of this study was to determine the lipolytic potential and the isoenzyme profile of fungi belonging to the Penicillium genus isolated from the Paranaense rainforest. Seven fungal strains were selected using qualitative screening. Quantitative analysis revealed that the isolate Penicillium sp. LBM 088 was the best producer of lipase, reaching 1224 U mL-1 of lipolytic activity. Zymogram gels of the seven selected strains showed different enzymatic profiles: In general, the molecular mass of proteins varied from 26 to 42 kDa. Also, proteins from fungi grown on olive oil showed a higher variation in their molecular mass than proteins from fungi grown without the oil. The search for new lipase-secreting organisms should lead to the exploitation of biodiversity in the region.

Enzymatic hydrolysis of barley straw for biofuel industry using a novel strain of Trametes villosa from Paranaense rainforest.

Agricultural practices generate lignocellulosic waste that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, fungal enzymes are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The aim of this work was to characterize LBM 033 strain and to analyze its efficiency in the hydrolysis of cellulosic substrates, including barley straw. LBM 033 strain was identified as Trametes villosa by molecular techniques, through the use of the ITS and rbp2 markers and the construction of phylogenetic trees. The cell-free supernatant of T. villosa LBM 033 showed high titers of hydrolytic enzymatic activities, necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing pure cellulose to cellobiose and glucose and also degraded the polysaccharides contained in barley straw to short soluble oligosaccharides. These results indicate that macro fungi from tropical soil environments, such as T. villosa LBM 033 can be a valuable resource for in-house, cost effective production of enzymes that can be applied in the hydrolysis stage, which could reduce the total cost of bioethanol production.

Adding value to lignocellulosic wastes via their use for endoxylanase production by Aspergillus fungi.

Agroforestry industries in the world generate lignocellulosic wastes that can be a huge problem of pollution, or the wastes can be used for different biotechonological applications such as substrates for microorganism growth and enzyme production. Fungi such as Aspergillus niger can grow in almost every substrate and produce hydrolytic enzymes such as endoxylanases, giving added value to agroforestry wastes generated by industries in the northeast of Argentina. In this context, the aim of this work was to use agroforestry wastes as substrates for the production of endoxylanases by Aspergillus niger and to optimize nitrogen sources and physical variables for the highest endoxylanase activity. A. niger LBM 055 and A. niger LBM 134 produced high endoxylanase levels when they were grown with sugarcane and cassava bagasses as carbon sources. A. niger LBM 134 reached the highest endoxylanase activity when nitrogen sources and physical variables were optimized. The fungus exhibited up to 110 U mL-1 of endoxylanase activity when it was grown with sugarcane bagasse and more than 160 U mL-1 with cassava bagasse. Therefore, endoxylanase production was optimized using agricultural bagasses and cost 20 times less than enzyme production using synthetic xylan.

Prevention of C5aR1 signaling delays microglial inflammatory polarization, favors clearance pathways and suppresses cognitive loss.

BACKGROUND: Pharmacologic inhibition of C5aR1, a receptor for the complement activation proinflammatory fragment, C5a, suppressed pathology and cognitive deficits in Alzheimer's disease (AD) mouse models. To validate that the effect of the antagonist was specifically via C5aR1 inhibition, mice lacking C5aR1 were generated and compared in behavior and pathology. In addition, since C5aR1 is primarily expressed on cells of the myeloid lineage, and only to a lesser extent on endothelial cells and neurons in brain, gene expression in microglia isolated from adult brain at multiple ages was compared across all genotypes. METHODS: C5aR1 knock out mice were crossed to the Arctic AD mouse model, and characterized for pathology and for behavior performance in a hippocampal dependent memory task. CX3CR1GFP and CCR2RFP reporter mice were bred to C5aR1 sufficient and knockout wild type and Arctic mice to enable sorting of microglia (GFP-positive, RFP-negative) isolated from adult brain at 2, 5, 7 and 10 months of age followed by RNA-seq analysis. RESULTS: A lack of C5aR1 prevented behavior deficits at 10 months, although amyloid plaque load was not altered. Immunohistochemical analysis showed no CCR2+ monocytes/macrophages near the plaques in the Arctic brain with or without C5aR1. Microglia were sorted from infiltrating monocytes (GFP and RFP-positive) for transcriptome analysis. RNA-seq analysis identified inflammation related genes as differentially expressed, with increased expression in the Arctic mice relative to wild type and decreased expression in the Arctic/C5aR1KO relative to Arctic. In addition, phagosomal-lysosomal gene expression was increased in the Arctic mice relative to wild type but further increased in the Arctic/C5aR1KO mice. A decrease in neuronal complexity was seen in hippocampus of 10 month old Arctic mice at the time that correlates with the behavior deficit, both of which were rescued in the Arctic/C5aR1KO. CONCLUSIONS: These data are consistent with microglial polarization in the absence of C5aR1 signaling reflecting decreased induction of inflammatory genes and enhancement of degradation/clearance pathways, which is accompanied by preservation of CA1 neuronal complexity and hippocampal dependent cognitive function. These results provide links between microglial responses and loss of cognitive performance and, combined with the previous pharmacological approach to inhibit C5aR1 signaling, support the potential of this receptor as a novel therapeutic target for AD in humans.

Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain.

BACKGROUND: The complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain. METHODS: To identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1+ neurons in both wild type mice and a mouse model of Alzheimer's disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis. RESULTS: While C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1+ neurons, the brains of C1qa FL/FL :Cx3cr1 CreERT2 mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 CreERT2/WganJ mice. C1q expression in C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice relative to controls, and C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice had minimal, if any, reduction in plasma C1q. CONCLUSIONS: Thus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 CreERT2/WganJ deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodegenerative disorders.

C5a Increases the Injury to Primary Neurons Elicited by Fibrillar Amyloid Beta.

C5aR1, the proinflammatory receptor for C5a, is expressed in the central nervous system on microglia, endothelial cells, and neurons. Previous work demonstrated that the C5aR1 antagonist, PMX205, decreased amyloid pathology and suppressed cognitive deficits in two Alzheimer's Disease (AD) mouse models. However, the cellular mechanisms of this protection have not been definitively demonstrated. Here, primary cultured mouse neurons treated with exogenous C5a show reproducible loss of MAP-2 staining in a dose-dependent manner within 24 hr of treatment, indicative of injury to neurons. This injury is prevented by the C5aR1 antagonist PMX53, a close analog of PMX205. Furthermore, primary neurons derived from C5aR1 null mice exhibited no MAP-2 loss after exposure to the highest concentration of C5a tested. Primary mouse neurons treated with both 100 nM C5a and 5 µM fibrillar amyloid beta (fAß), to model what occurs in the AD brain, showed increased MAP-2 loss relative to either C5a or fAß alone. Blocking C5aR1 with PMX53 (100 nM) blocked the loss of MAP2 in these primary neurons to the level seen with fAß alone. Similar experiments with primary neurons derived from C5aR1 null mice showed a loss of MAP-2 due to fAß treatment. However, the addition of C5a to the cultures did not enhance the loss of MAP-2 and the addition of PMX53 to the cultures did not change the MAP-2 loss in response to fAß. Thus, at least part of the beneficial effects of C5aR1 antagonist in AD mouse models may be due to protection of neurons from the toxic effects of C5a.

Analysis of the Putative Role of CR1 in Alzheimer's Disease: Genetic Association, Expression and Function.

Chronic activation of the complement system and induced inflammation are associated with neuropathology in Alzheimer's disease (AD). Recent large genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the C3b/C4b receptor (CR1 or CD35) that are associated with late onset AD. Here, anti-CR1 antibodies (Abs) directed against different epitopes of the receptor, were used to localize CR1 in brain, and relative binding affinities of the CR1 ligands, C1q and C3b, were assessed by ELISA. Most Abs tested stained red blood cells in blood vessels but showed no staining in brain parenchyma. However, two monoclonal anti-CR1 Abs labeled astrocytes in all of the cases tested, and this reactivity was preabsorbed by purified recombinant human CR1. Human brain-derived astrocyte cultures were also reactive with both mAbs. The amount of astrocyte staining varied among the samples, but no consistent difference was conferred by diagnosis or the GWAS-identified SNPs rs4844609 or rs6656401. Plasma levels of soluble CR1 did not correlate with diagnosis but a slight increase was observed with rs4844609 and rs6656401 SNP. There was also a modest but statistically significant increase in relative binding activity of C1q to CR1 with the rs4844609 SNP compared to CR1 without the SNP, and of C3b to CR1 in the CR1 genotypes containing the rs6656401 SNP (also associated with the larger isoform of CR1) regardless of clinical diagnosis. These results suggest that it is unlikely that astrocyte CR1 expression levels or C1q or C3b binding activity are the cause of the GWAS identified association of CR1 variants with AD. Further careful functional studies are needed to determine if the variant-dictated number of CR1 expressed on red blood cells contributes to the role of this receptor in the progression of AD, or if another mechanism is involved.

Complement activation fragment C5a receptors, CD88 and C5L2, are associated with neurofibrillary pathology.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative dementia characterized by the decline of cognition and the presence of neuropathological changes including neuronal loss, neurofibrillary pathology and extracellular senile plaques. A neuroinflammatory process is also triggered and complement activation has been hypothesized to have a relevant role in this local inflammatory response. C5a, a proinflammatory anaphylatoxin generated after complement activation, exerts its chemotactic and inflammatory functions through the CD88 receptor while the more recently discovered C5L2 receptor has been postulated to have an anti-inflammatory role. Previously, we reported that a CD88 specific antagonist (PMX205) decreased the pathology and improved cognition in transgenic models of AD suggesting that C5a/C5aR interaction has an important role in the progression of the disease. METHODS: The present study characterizes the expression of the two receptors for C5a in human brain with confirmed post mortem diagnosis of vascular dementia (VD) or AD as well as age matched controls by immunohistochemistry and Western blot analysis using several antibodies against different epitopes of the human receptors. RESULTS: The CD88 and C5L2 antibodies revealed increased expression of both receptors in AD samples as compared to age-matched controls or VD brain tissue by Western blot and immunohistochemistry, using multiple antibodies and distinct cohorts of brain tissue. Immunostaining showed that both the C5L2 and CD88 antibodies similarly labeled abundant neurofibrillary tangles, neuropil threads and dystrophic neurites associated with plaques in the hippocampus and frontal cortex of AD cases. In contrast, little or no neuronal staining, tangles or dystrophic neurites associated with plaques were observed in control or VD brains. CD88 and C5L2 receptors are associated with both early (AT8) and mature (PHF1) neurofibrillary tangles and can be found either independently or colocalized with each other. CONCLUSIONS: The observed association of CD88 and C5L2 with neurofibrillary pathology suggests a common altered pathway of degradation.

Contribution of complement activation pathways to neuropathology differs among mouse models of Alzheimer's disease.

BACKGROUND: Complement proteins and activation products have been found associated with neuropathology in Alzheimer's disease (AD). Recently, a C5a receptor antagonist was shown to suppress neuropathology in two murine models of AD, Tg2576 and 3xTg. Previously, a genetic deficiency of C1q in the Tg2576 mouse model showed an accumulation of fibrillar plaques similar to the complement sufficient Tg2576, but reactive glia were significantly decreased and neuronal integrity was improved suggesting detrimental consequences for complement activation in AD. The goal of this study was to define the role of the classical complement activation pathway in the progression of pathology in the 3xTg mouse that develops tangles in addition to fibrillar plaques (more closely reflecting human AD pathology) and to assess the influence of complement in a model of AD with a higher level of complement hemolytic activity. METHODS: 3xTg mice deficient in C1q (3xTgQ-/-) were generated, and both 3xTg and 3xTgQ-/- were backcrossed to the BUB mouse strain which has higher in vitro hemolytic complement activity. Mice were aged and perfused, and brain sections stained for pathological markers or analyzed for proinflammatory marker expression. RESULTS: 3xTgQ-/- mice showed similar amounts of fibrillar amyloid, reactive glia and hyperphosphorylated tau as the C1q-sufficient 3xTg at the ages analyzed. However, 3xTg and 3xTgQ-/- on the BUB background developed pathology earlier than on the original 3xTg background, although the presence of C1q had no effect on neuropathological and pro-inflammatory markers. In contrast to that seen in other transgenic models of AD, C1q, C4 and C3 immunoreactivity was undetectable on the plaques of 3xTg in any background, although C3 was associated with reactive astrocytes surrounding the plaques. Importantly, properdin a component of the alternative complement pathway was associated with plaques in all models. CONCLUSIONS: In contrast to previously investigated transgenic models of AD, development of neuropathology in 3xTg mice, which progresses much slower than other murine models, may not be influenced by fibrillar amyloid mediated activation of the classical complement pathway, suggesting that the alternative complement pathway activation or a C3-independent cleavage of C5 could account for the detrimental effects in these mice that are prevented by the C5a receptor antagonist. Furthermore, the paucity of complement activation may be a factor in the slower kinetics of progression of pathology in the 3xTg model of this disease.

White rot fungi laccases for biotechnological applications.

White rot fungi have an enzymatic system producing oxidative and hydrolytic enzymes that act on the degradation of certain components of the cell wall. They can be applied in several technological processes, such as paper industry, bio-fuels and environmental pollution. Laccases are multi-copper enzymes of wide substrate specificity and high non-specific oxidation capacity that use molecular oxygen to oxidize various aromatic compounds, and are highly relevant biotechnological applications. In this review, we present some significant patents on laccase production and recombinant DNA technology for diverse biotechnology applications.

Microglial C5aR (CD88) expression correlates with amyloid-beta deposition in murine models of Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by the accumulation of amyloid-beta protein and neuronal loss, is the leading cause of age-related dementia in the world today. The disease is also associated with neuroinflammation, robust activation of astrocytes and microglia, and evidence of activation of the complement system, localized with both fibrillar amyloid-beta (fAbeta) plaques and tangles. The observations are consistent with a complement-dependent component of AD progression. We have previously shown that inhibition of the major complement receptor for C5a (CD88) with the antagonist PMX205 results in a significant reduction in pathology in two mouse models of AD. To further characterize the role of complement in AD-related neuroinflammation, we examined the age- and disease-associated expression of CD88 in brain of transgenic mouse models of AD and the influence of PMX205 on the presence of various complement activation products using flow cytometry, western blot, and immunohistochemistry. CD88 was found to be up-regulated in microglia, in the immediate vicinity of amyloid plaques. While thioflavine plaque load and glial recruitment is significantly reduced after treatment with PMX205, C1q remains co-localized with fAbeta plaques and C3 is still expressed by the recruited astrocytes. Thus, with PMX205, potentially beneficial activities of these early complement components may remain intact, while detrimental activities resulting from C5a-CD88 interaction are inhibited. This further supports the targeted inhibition of specific complement mediated activities as an approach for AD therapy.

Copper inducing effect on laccase production of white rot fungi native from Misiones (Argentina).

Fungi may be selected as models for gene expression studies and further adaptation for biotechnological enzyme production. The aim of this work was to evaluate laccase production and to analyze the effect of Cu(2+) on selected fungi natives of Misiones, Ganoderma applanatum (strain F), Peniophora sp. (BAFC 633), Pycnoporus sanguineus (BAFC 2126) and Coriolus versicolor f. antarcticus (BAFC 266). Fungi secretion system of G. applanatum, Peniophora sp., P. sanguineus and C. versicolor f. antarcticus is sensitive to stimulation by copper. Biomass values of G. applanatum, Peniophora sp. and C. versicolor f. antarcticus did not show differences between treatments. P. sanguineus biomass underwent a dramatic growth inhibition with 1mM Cu(2+) and marked delay in growth with 0.5mM Cu(2+). Proteins were increased with copper in Peniophora sp., C. versicolor and G. applanatum. G. applanatum and Peniophora sp. reached the highest enzyme activity at 10th day equivalent to 49.2-fold and 19.7-fold higher than the control samples, respectively. Copper produced an increase of constitutive laccases in all fungi and an additional inducible isoenzyme in Peniophora sp., C. versicolor f. antarcticus and G. applanatum.

Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related dementia, characterized by amyloid plaques, neurofibrillary tangles, neuroinflammation, and neuronal loss in the brain. Components of the complement system, known to produce a local inflammatory reaction, are associated with the plaques and tangles in AD brain, and thus a role for complement-mediated inflammation in the acceleration or progression of disease has been proposed. A complement activation product, C5a, is known to recruit and activate microglia and astrocytes in vitro by activation of a G protein-coupled cell-surface C5aR. Here, oral delivery of a cyclic hexapeptide C5a receptor antagonist (PMX205) for 2-3 mo resulted in substantial reduction of pathological markers such as fibrillar amyloid deposits (49-62%) and activated glia (42-68%) in two mouse models of AD. The reduction in pathology was correlated with improvements in a passive avoidance behavioral task in Tg2576 mice. In 3xTg mice, PMX205 also significantly reduced hyperphosphorylated tau (69%). These data provide the first evidence that inhibition of a proinflammatory receptor-mediated function of the complement cascade (i.e., C5aR) can interfere with neuroinflammation and neurodegeneration in AD rodent models, suggesting a novel therapeutic target for reducing pathology and improving cognitive function in human AD patients.

Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease resulting in progressive cognitive decline. Amyloid plaque deposits consisting specifically of beta-amyloid peptides that have formed fibrils displaying beta-pleated sheet conformation are associated with activated microglia and astrocytes, are colocalized with C1q and other complement activation products, and appear at the time of cognitive decline in AD. Amyloid precursor protein (APP) transgenic mouse models of AD that lack the ability to activate the classical complement pathway display less neuropathology than do the APPQ+/+ mice, consistent with the hypothesis that complement activation and the resultant inflammation may play a role in the pathogenesis of AD. Further investigation of the presence of complement proteins C3 and C4 in the brain of these mice demonstrate that both C3 and C4 deposition increase with age in APPQ+/+ transgenic mice, as expected with the age-dependent increase in fibrillar beta-amyloid deposition. In addition, while C4 is predominantly localized on the plaques and/or associated with oligodendrocytes in APPQ+/+ mice, little C4 is detected in APPQ-/- brains consistent with a lack of classical complement pathway activation because of the absence of C1q in these mice. In contrast, plaque and cell associated C3 immunoreactivity is seen in both animal models and, surprisingly, is higher in APPQ-/- than in APPQ+/+ mice, providing evidence for alternative pathway activation. The unexpected increase in C3 levels in the APPQ-/- mice coincident with decreased neuropathology provides support for the hypothesis that complement can mediate protective events as well as detrimental events in this disease. Finally, induced expression of C3 in a subset of astrocytes suggests the existence of differential activation states of these cells.

The double-edged flower: roles of complement protein C1q in neurodegenerative diseases.

A role for the complement cascade in AD neuropathology was hypothesized over a decade ago, and the results of a significant number of in vitro studies are consistent with the involvement of this pathway in AD pathogenesis (reviewed in). Since C1q is colocalized with thioflavine-positive plaques and the C5b-9 complement membrane attack complex is detected in AD brain at autopsy, it is reasonable to hypothesize that complement activation has a role in the manifestation of AD either by its lytic capacity or as a trigger of glial infiltration and initiation of potentially damaging inflammation. The observed diminished glial activation and reduced loss of neuronal integrity in a murine model overexpressing mutant human APP but lacking the ability to activate the classical complement cascade provide the first direct evidence for a detrimental role of C1q, and presumably activation of the classical complement pathway in an animal model of AD. Research is now focused on generating mouse models that more closely mimic the human disease, so that the role of complement activation and inflammation on the behavioral/learning and memory dysfunction that occurs in this disease can be assessed. In addition, candidate therapies such as targeted inhibition of complement activation will need to be tested in these animal models as a step toward treatment of humans with the disease. However, it is important that the potential for a protective effect of C1q early on in disease progression should not be overlooked. Rather, strategies that enhance or mimic the protective effects of C1q as well as strategies that inhibit the detrimental processes should be fully investigated.