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Meniscal lesions in vascularized regions are known to regenerate while lack of vascular supply leads to poor healing. Here, we developed and validated a novel methodology for three-dimensional structural analysis of meniscal vascular structures with high-resolution microcomputed tomography (µCT). We collected porcine medial menisci from 10 neonatal (not-developed meniscus, n-) and 10 adults (fully developed meniscus, a-). The menisci were cut into anatomical regions (anterior horn (n-AH and a-AH), central body (n-CB and a-CB), and posterior horn (n-PH and a-PH). Specimens were cut in half, fixed, and one specimen underwent critical point drying and µCT imaging, while other specimen underwent immunohistochemistry and vascularity biomarker CD31 staining for validation of µCT. Parameters describing vascular structures were calculated from µCT. The vascular network in neonatal spread throughout meniscus, while in adult was limited to a few vessels in outer region, mostly on femoral side. n-AH, n-CB, and n-PH had 20, 17, and 11 times greater vascular volume fraction than adult, respectively. Moreover, thickness of blood vessels, in three regions, was six times higher in adults than in neonatal. a-PH appeared to have higher vascular fraction, longer and thicker blood vessels than both a-AH and a-CB. Overall, neonatal regions had a higher number of blood vessels, more branching, and higher tortuosity compared to adult regions. For the first time, critical point drying-based µCT imaging allowed detailed three-dimensional visualization and quantitative analysis of vascularized meniscal structures. We showed more vascularity in neonatal menisci, while adult menisci had fewer and thicker vascularity especially limited to the femoral surface., (© 2024 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

The nutritional, antimicrobial, and antioxidant properties of bovine colostrum (BC) have encouraged its use in animal nutrition as a functional food in recent years. Nonetheless, the potential implications of BC supplementation on meat quality remain to be thoroughly assessed. To address this, thirty-nine New Zealand White rabbits (n = 13/group) were fed different dietary regimens until slaughter.: commercial standard diet for the control group (C) and C with 2.5% and 5% w/w of BC for BC-2.5 and BC-5 groups, respectively. Rabbits were slaughtered at 91 days of age and meat quality, and sensory characteristics were evaluated at days 2 (48 h after slaughter), 5, and 10 of refrigerated storage at 4 °C. The addition of colostrum in the diet resulted in a reduction of the total viable count, albeit only at the highest concentration and at the final detection, whereas for Lactobacillus spp. and Pseudomonas spp., there was little or no effect. The colour coordinates showed no differences between the groups, but they varied over time according to diet. Some differences between groups emerged in the definition of sensory attributes but did not affect the overall liking and overall scores of individual descriptors. These results indicate that the use of colostrum in rabbit feeding does not significantly impart meat quality and sensory attributes, but the potential of this valuable by-product for the food industry needs further investigation., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in this article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The permeability of the immature intestine is higher in newborns than in adults; a damaged gut barrier in young animals increases the susceptibility to digestive and infectious diseases later in life. It is therefore of major importance to avoid impairment of the intestinal barrier, specifically in a delicate phase of development, such as weaning. This study aimed to evaluate the effects of bovine colostrum supplementation on the intestinal barrier, such as the intestinal morphology and proliferation level and tight junctions expression (zonulin) and enteric nervous system (ENS) inflammation status (through the expression of PGP9.5 and GFAP) in fattening rabbits. Rabbits of 35 days of age were randomly divided into three groups (n = 13) based on the dietary administration: commercial feed (control group, CTR) and commercial feed supplemented with 2.5% and 5% bovine colostrum (BC1 and BC2 groups, respectively). Rabbits receiving the BC1 diet showed a tendency to have better duodenum morphology and higher proliferation rates ( p < 0.001) than the control group. An evaluation of the zonulin expression showed that it was higher in the BC2 group, suggesting increased permeability, which was partially confirmed by the expression of GFAP. Our results suggest that adding 2.5% BC into the diet could be a good compromise between intestinal morphology and permeability, since rabbits fed the highest inclusion level of BC showed signs of higher intestinal permeability.

This study evaluates the morpho-functional modifications that characterize meniscal development from neonatal to adult dogs. Even if menisci are recognized as essential structures for the knee joint, poor information is available about their morphogenesis, in particular in dog models. Menisci from a group of Dobermann Pinchers aged 0, 10, 30 days, and 4 years (T0, T10, T30, adult, respectively) were analyzed by SEM, histochemistry (Safranin O and Picro Sirius Red Staining analyzed under a polarized light microscope), immunofluorescences (collagen type I and II), biomechanical (compression) and biochemical analyses (glycosaminoglycans, GAGs, and DNA content). SEM analyses revealed that the T0 meniscus is a bulgy structure that during growth tends to flatten, firstly in the inner zone (T10) and then even in the outer zone (T30), until the achievement of the completely smooth adult final shape. These results were further supported by the histochemistry analyses in which the deposition of GAGs started from T30, and the presence of type I birefringent collagen fibers was observed from T0 to T30, while poorly refringent type III collagen fibers were observed in the adult dogs. Double immunofluorescence analyses also evidenced that the neonatal meniscus contains mainly type I collagen fibers, as well as the T10 meniscus, and demonstrated a more evident regionalization and crimping in the T30 and adult meniscus. Young's elastic modulus of the meniscus in T0 and T10 animals was lower than the T30 animals, and this last group was also lower than adult ones (T0-T10 vs T30 vs adult). Biochemical analysis confirmed that cellularity decreases over time from neonatal to adult (p < 0.01). The same decreasing trend was observed in GAGs deposition. These results may suggest that the postnatal development of canine meniscus may be related to the progressive functional locomotory development: after birth, the meniscus acquires its functionality over time, through movement, load, and growth itself., 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 © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The availability and cost of fishmeal constitute a bottleneck in Atlantic salmon production expansion. Fishmeal is produced from wild fish species and constitutes the major feed ingredient in carnivorous species such as the Atlantic salmon. These natural stocks are at risk of depletion and it is therefore of major importance to find alternative protein sources that meet the nutritional requirements of the Atlantic salmon, without compromising the animals' health. Terrestrial animal by-products have been used in aquaculture feed, but their use is limited by the lack of several essential amino acids and consumer acceptance. In the case of plant ingredients, it is necessary to take into account both their concentration and the extraction methodologies, since, if not dosed correctly, they can cause macro- and microscopic alterations of the structure of the gastrointestinal tract and can also negatively modulate the microbiota composition. These alterations may compromise the digestive functions, growth of the animal, and, ultimately, its well-being. An updated revision of alternative protein sources is provided, with the respective impact on the intestine health in terms of both morphology and microbiota composition. Such information may constitute the premise for the choice and development of Atlantic salmon feeds that guarantee fish health and growth performance without having a significant impact on the surrounding environment, both in terms of depletion of the fish's natural stocks and in terms of pressure on the terrestrial agriculture. The sustainability of aquaculture should be a priority when choosing next-generation ingredients.

According to statistics and future predictions, meat consumption will increase in the coming years. Considering both the environmental impact of intensive livestock farming and the importance of protecting animal welfare, the necessity of finding alternative strategies to satisfy the growing meat demand is compelling. Biotechnologies are responding to this demand by developing new strategies for producing meat in vitro. The manufacturing of cultured meat has faced criticism concerning, above all, the practical issues of culturing together different cell types typical of meat that are partly responsible for meat's organoleptic characteristics. Indeed, the existence of a cross talk between adipose and muscle cells has critical effects on the outcome of the co-culture, leading to a general inhibition of myogenesis in favor of adipogenic differentiation. This review aims to clarify the main mechanisms and the key molecules involved in this cross talk and provide an overview of the most recent and successful meat culture 3D strategies for overcoming this challenge, focusing on the approaches based on farm-animal-derived cells.

The success of cell-based approaches for the treatment of cartilage or fibro-cartilaginous tissue defects requires an optimal cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. For this purpose, the aim of this study was to evaluate the use of endostatin (COL18A1), an anti-angiogenic factor, which is physiologically involved in cell differentiation during meniscus development. Swine neonatal meniscal cells not yet subjected to mechanical stimuli were extracted, cultured in fibrin hydrogel scaffolds, and treated at two different time points (T1 = 9 days and T2 = 21 days) with different concentrations of COL18A1 (10 ng/mL; 100 ng/mL; 200 ng/mL). At the end of the treatments, the scaffolds were examined through biochemical, molecular, and histochemical analyses. The results showed that the higher concentration of COL18A1 promotes a fibro-chondrogenic phenotype and improves cellularity index (DNA content, p < 0.001) and cell efficiency (GAGs/DNA ratio, p < 0.01) after 21 days. These data are supported by the molecular analysis of collagen type I (COL1A1, a marker of fibrous-like tissue, p < 0.001), collagen type II (COL2A1, a marker of cartilaginous-like tissue, p < 0.001) and SRY-Box Transcription Factor 9 (SOX9, an early marker of chondrogenicity, p < 0.001), as well as by histological analysis (Safranin-O staining), laying the foundations for future studies evaluating the involvement of 3D endostatin hydrogel scaffolds in the differentiation of avascular tissues.

Menisci play an essential role in shock absorption, joint stability, load resistance and its transmission thanks to their conformation. Adult menisci can be divided in three zones based on the vascularization: an avascular inner zone with no blood supply, a fully vascularized outer zone, and an intermediate zone. This organization, in addition to the incomplete knowledge about meniscal biology, composition, and gene expression, makes meniscal regeneration still one of the major challenges both in orthopedics and in tissue engineering. To overcome this issue, we aimed to investigate the role of hypoxia in the differentiation of the three anatomical areas of newborn piglet menisci (anterior horn (A), central body (C), and posterior horn (P)) and its effects on vascular factors. After sample collection, menisci were divided in A, C, P, and they were cultured in vitro under hypoxic (1% O 2 ) and normoxic (21% O 2 ) conditions at four different experimental time points (T0 = day of explant; T7 = day 7; T10 = day 10; T14 = day 14); samples were then evaluated through immune, histological, and molecular analyses, cell morpho-functional characteristics; with particular focus on matrix composition and expression of vascular factors. It was observed that hypoxia retained the initial phenotype of cells and induced extracellular matrix production resembling a mature tissue. Hypoxia also modulated the expression of angiogenic factors, especially in the early phase of the study. Thus, we observed that hypoxia contributes to the fibro-chondrogenic differentiation with the involvement of angiogenic factors, especially in the posterior horn, which corresponds to the predominant weight-bearing portion.

Aluminum based adjuvants are widely used in commercial vaccines, since they are known to be safe and effective with a variety of antigens. The effect of antigen adsorption onto Aluminum Hydroxide is a complex area, since several mechanisms are involved simultaneously, whose impact is both antigen and formulation conditions dependent. Moreover, the mode of action of Aluminum Hydroxide is itself complex, with many mechanisms operating simultaneously. Within the literature there are contrasting theories regarding the effect of adsorption on antigen integrity and stability, with reports of antigen being stabilized by adsorption onto Aluminum Hydroxide, but also with contrary reports of antigen being destabilized. With the aim to understand the impact of adsorption on three recombinant proteins which, following in vivo immunization, are able to induce functional bactericidal antibodies against Neisseria meningitidis type B, we used a range of physico-chemical tools, such as DSC and UPLC, along with in vitro binding of antibodies that recognize structural elements of the proteins, and supported the in vitro data with in vivo evaluation in mice studies. We showed that, following exposure to accelerated degradation conditions involving heat, the recombinant proteins, although robust, were stabilized by adsorption onto Aluminum Hydroxide and retain their structural integrity unlike the not adsorbed proteins. The measure of the Melting Temperature was a useful tool to compare the behavior of proteins adsorbed and not adsorbed on Aluminum Hydroxide and to predict protein stability., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was sponsored by Novartis Vaccines and Diagnostics Srl (now part of the GSK group of companies) which was involved in all stages of the study conduct and analysis. AC, SS, FF, BB, MU, MP and DOH were employees of Novartis Vaccines and Diagnostics Srl at the time of the study (now part of the GSK group of companies). AC, SS, FF, BB and DOH are now employees of the GSK group of companies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus., (Copyright © 2016 Fiaschi et al.)

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.

Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The sleep disorder narcolepsy is linked to the HLA-DQB1*0602 haplotype and dysregulation of the hypocretin ligand-hypocretin receptor pathway. Narcolepsy was associated with Pandemrix vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic. In contrast, very few cases were reported after Focetria vaccination (a differently manufactured adjuvanted influenza pandemic vaccine). We hypothesized that differences between these vaccines (which are derived from inactivated influenza viral proteins) explain the association of narcolepsy with Pandemrix-vaccinated subjects. A mimic peptide was identified from a surface-exposed region of influenza nucleoprotein A that shared protein residues in common with a fragment of the first extracellular domain of hypocretin receptor 2. A significant proportion of sera from HLA-DQB1*0602 haplotype-positive narcoleptic Finnish patients with a history of Pandemrix vaccination (vaccine-associated narcolepsy) contained antibodies to hypocretin receptor 2 compared to sera from nonnarcoleptic individuals with either 2009 A(H1N1) pandemic influenza infection or history of Focetria vaccination. Antibodies from vaccine-associated narcolepsy sera cross-reacted with both influenza nucleoprotein and hypocretin receptor 2, which was demonstrated by competitive binding using 21-mer peptide (containing the identified nucleoprotein mimic) and 55-mer recombinant peptide (first extracellular domain of hypocretin receptor 2) on cell lines expressing human hypocretin receptor 2. Mass spectrometry indicated that relative to Pandemrix, Focetria contained 72.7% less influenza nucleoprotein. In accord, no durable antibody responses to nucleoprotein were detected in sera from Focetria-vaccinated nonnarcoleptic subjects. Thus, differences in vaccine nucleoprotein content and respective immune response may explain the narcolepsy association with Pandemrix., (Copyright © 2015, American Association for the Advancement of Science.)

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.

Vaccines are the most effective agents to control infections. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance protective immune responses. However, the molecular mechanism of action of most adjuvants is ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freund's adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is always associated with a weak transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freund's adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination.

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.

The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.

Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway.

This study evaluated the feasibility of using γ-irradiation for preparing sterile poly(lactide-co-glycolide) (PLG) formulations for vaccines. PLG microparticles were prepared by water-in-oil-in-water double-emulsion technique and lyophilized. The vials were γ-irradiated for sterilization process. Antigens from Neisseria meningitidis were adsorbed onto the surface of the particles and were characterized for protein adsorption. Antigens adsorbed onto the surface of the irradiated particles within 30 min. Mice were immunized with these formulations, and vaccine potency was measured as serum bactericidal titers. The γ-irradiated PLG particles resulted in equivalent serum bactericidal titers against a panel of five N. meningitidis strains as the nonirradiated PLG particles. The use of PLG polymers with different molecular weights did not influence the vaccine potency. The PLG particles prepared by γ-irradiation of the lyophilized formulations replace the need for aseptic manufacturing of vaccine formulations. This approach may enable the use of PLG formulations with a variety of antigens and stockpiling for pandemics., (Copyright © 2010 Wiley-Liss, Inc.)

Different signal-regulated serine/threonine kinases phosphorylate class II histone deacetylases (HDACs) to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme C alpha, A alpha, B/PR55 alpha. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid or RNA interference have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import.

Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation.

Histone deacetylases (HDACs) and histone acetyl transferases (HATs) are two counteracting enzyme families whose enzymatic activity controls the acetylation state of protein lysine residues, notably those contained in the N-terminal extensions of the core histones. Acetylation of histones affects gene expression through its influence on chromatin conformation. In addition, several non-histone proteins are regulated in their stability or biological function by the acetylation state of specific lysine residues. HDACs intervene in a multitude of biological processes and are part of a multiprotein family in which each member has its specialized functions. In addition, HDAC activity is tightly controlled through targeted recruitment, protein-protein interactions and post-translational modifications. Control of cell cycle progression, cell survival and differentiation are among the most important roles of these enzymes. Since these processes are affected by malignant transformation, HDAC inhibitors were developed as antineoplastic drugs and are showing encouraging efficacy in cancer patients.

The aim of this study was to investigate the mechanism of activation of human heparanase, a key player in heparan sulfate degradation, thought to be involved in normal and pathologic cell migration processes. Active heparanase arises as a product of a series of proteolytic processing events. Upon removal of the signal peptide, the resulting, poorly active 65 kDa species undergoes the excision of an intervening 6 kDa fragment generating an 8 kDa polypeptide and a 50 kDa polypeptide, forming the fully active heterodimer. By engineering of tobacco etch virus protease cleavage sites at the N- and C-terminal junctions of the 6 kDa fragment, we were able to reproduce the proteolytic activation of heparanase in vitro using purified components, showing that cleavage at both sites leads to activation in the absence of additional factors. On the basis of multiple-sequence alignment of the N-terminal fragment, we conclude that the first beta/alpha/beta element of the postulated TIM barrel fold is contributed by the 8 kDa subunit and that the excised 6 kDa fragment connects the second beta-strand and the second alpha-helix of the barrel. Substituting the 6 kDa fragment with the topologically equivalent loop from Hirudinaria manillensis hyaluronidase or connecting the 8 and 50 kDa fragments with a spacer of three glycine-serine pairs resulted in constitutively active, single-chain heparanases which were comparable to the processed, heterodimeric enzyme with regard to specific activity, chromatographic profile of hydrolysis products, complete inhibition at NaCl concentrations above 600 mM, a pH optimum of pH approximately 5, and inhibition by heparin with IC(50)s of 0.9-1.5 ng/microL. We conclude that (1) the heparanase heterodimer (alpha/beta)(8)-TIM barrel fold is contributed by both 8 and 50 kDa subunits with the 6 kDa connecting fragment leading to inhibition of heparanase by possibly obstructing access to the active site, (2) proteolytic excision of the 6 kDa fragment is necessary and sufficient for heparanase activation, and (3) our findings open the way to the production of recombinant, constitutively active single-chain heparanase for structural studies and for the identification of inhibitors.

The present work reports a mass spectrometric investigation of the NS2/3 protein, a protease from hepatitis C virus (HCV). During routine protein manipulation, in the presence of 100 mM beta-mercaptoethanol and under denatured conditions, the protein was unexpectedly modified at its cysteine residues, and the increased molecular weight corresponded to one molecule of beta-mercaptoethanol bound. The modified protein, once refolded, was found to be less active than the unmodified one. The aim of this work was to investigate whether the reactivity of cysteines with beta-mercaptoethanol involves one specific, highly reactive residue of the sequence, or if the modification is a random process. Liquid chromatography (LC) coupled on-line with an electrospray ion trap mass spectrometer was used to identify the modification sites. It was found that five cysteines out of nine had reacted with beta-mercaptoethanol, none of them showing a significantly higher reactivity than the others. 95% of sequence coverage was obtained., (Copyright 2002 John Wiley & Sons, Ltd.)

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Maturational cleavage of the hepatitis C virus polyprotein involves the viral chymotrypsin-like serine protease NS3. The substrate binding site of this enzyme is unusually flat and featureless. We here show that NS3 has a highly asymmetric charge distribution that is characterized by strong positive potentials in the vicinity of its active site and in the S5/S6 region. Using electrostatic potential calculations, we identified determinants of this positive potential, and the role of six different residues was explored by site-directed mutagenesis. Mutation of residues in the vicinity of the active site led to changes in k(cat) values of a peptide substrate indicating that basic amino acids play a role in the stabilization of the transition state. Charge neutralization in the S5/S6 region increased the K(m) values of peptide substrates in a manner that depended on the presence of negatively charged residues in the P5 and P6 positions. K(i) values of hexapeptide acids spanning P6-P1 (product inhibitors) were affected by charge neutralization in both the active site region and the S5/S6 region. Pre-steady-state kinetic data showed that the electrostatic surface potential is used by this enzyme to enhance collision rates between peptidic ligands and the active site. Calculations of the interaction energies of protease-substrate or protease-inhibitor complexes showed that electrostatic interaction energies oppose the formation of a tightly bound complex due to an unfavorable change in the desolvation energy. We propose that desolvation costs are minimized by avoiding the formation of individual ion pair interactions through the use of clusters of positively charged residues in the generation of local electrostatic potentials.

Previously, this laboratory identified clusters of alpha-, beta-, and mast cell protease-7-like tryptase genes on human chromosome 16p13.3. The present work characterizes adjacent genes encoding novel serine proteases, termed gamma-tryptases, and generates a refined map of the multitryptase locus. Each gamma gene lies between an alpha1H Ca2+ channel gene (CACNA1H) and a betaII- or betaIII-tryptase gene and is approximately 30 kb from polymorphic minisatellite MS205. The tryptase locus also contains at least four tryptase-like pseudogenes, including mastin, a gene expressed in dogs but not in humans. Genomic DNA blotting results suggest that gammaI- and gammaII-tryptases are alleles at the same site. betaII- and betaIII-tryptases appear to be alleles at a neighboring site, and alphaII- and betaI-tryptases appear to be alleles at a third site. gamma-Tryptases are transcribed in lung, intestine, and in several other tissues and in a mast cell line (HMC-1) that also expresses gamma-tryptase protein. Immunohistochemical analysis suggests that gamma-tryptase is expressed by airway mast cells. gamma-Tryptase catalytic domains are approximately 48% identical with those of known mast cell tryptases and possess mouse homologues. We predict that gamma-tryptases are glycosylated oligomers with tryptic substrate specificity and a distinct mode of activation. A feature not found in described tryptases is a C-terminal hydrophobic domain, which may be a membrane anchor. Although the catalytic domains contain tryptase-like features, the hydrophobic segment and intron-exon organization are more closely related to another recently described protease, prostasin. In summary, this work describes gamma-tryptases, which are novel members of chromosome 16p tryptase/prostasin gene families. Their unique features suggest possibly novel functions.

A partial cDNA encoding bovine tryptase, an oligomeric serine proteinase previously isolated from bovine mast cells, was obtained by reverse transcription/polymerase chain reaction of mast cell mRNA, using combinations of primers designed on the basis of information obtained from partial sequencing of the purified protein. The complete amino acid sequence of bovine tryptase (245 residues) was deduced from a 711-bp nucleotide sequence and from Edman degradation of the protein. Bovine tryptase primary structure has an identity of about 75% with tryptases from other species and includes all the essential residues of the active-site regions; sequence data in the region of the putative substrate binding pocket suggest a rearrangement capable of maintaining the specificity of trypsin-like proteinases. From the same mast cell mRNA, cDNA encoding bovine trypsin protease inhibitor (BPTI) was obtained and amplified with specific primers, confirming the synthesis of BPTI in these cells. Results are consistent with previous data on the presence of BPTI and bovine tryptase in the same granules of bovine mast cells and with their interaction in vitro.