Crystal structure of human peroxiredoxin 5, a novel type of mammalian peroxiredoxin at 1.5 A resolution.

The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules such as thioredoxin, glutathione, trypanothione and AhpF. Peroxiredoxins have been identified in prokaryotes as well as in eukaryotes. Peroxiredoxin 5 (PRDX5) is a novel type of mammalian thioredoxin peroxidase widely expressed in tissues and located cellularly to mitochondria, peroxisomes and cytosol. Functionally, PRDX5 has been implicated in antioxidant protective mechanisms as well as in signal transduction in cells. We report here the 1.5 A resolution crystal structure of human PRDX5 in its reduced form. The crystal structure reveals that PRDX5 presents a thioredoxin-like domain. Interestingly, the crystal structure shows also that PRDX5 does not form a dimer like other mammalian members of the peroxiredoxin family. In the reduced form of PRDX5, Cys47 and Cys151 are distant of 13.8 A although these two cysteine residues are thought to be involved in peroxide reductase activity by forming an intramolecular disulfide intermediate in the oxidized enzyme. These data suggest that the enzyme would necessitate a conformational change to form a disulfide bond between catalytic Cys47 and Cys151 upon oxidation according to proposed peroxide reduction mechanisms. Moreover, the presence of a benzoate ion, a hydroxyl radical scavenger, was noted close to the active-site pocket. The possible role of benzoate in the antioxidant activity of PRDX5 is discussed.

Peroxiredoxin 5 expression in the human thyroid gland.

Peroxiredoxin 5 (PRDX5) is a newly discovered thioredoxin peroxidase able to reduce peroxides that is implicated in antioxidant protective mechanisms. We report here its expression in the human thyroid gland. Twenty-seven human thyroid specimens were examined by immunohistochemistry. They included six normal thyroid tissues, five multinodular goiters, nine hot nodules, two Hürthle cell adenomas, and five thyroids from patients with Graves' disease. In the control tissue, PRDX5 expression was heterogeneous, being stronger in cubical functionally active follicular cells than in flat quiescent thyrocytes. It was diffuse in the cytoplasm, occasionally localized in inclusions that most likely corresponded to mitochondria. This feature was particularly marked in the Hürthle cell adenoma case. In multinodular goiters, hot nodules, and Graves' thyroids, the cytosolic labeling was enhanced compared to the control tissue and a signal was also detected in few nuclei. To determine whether the level of expression was different between multinodular goiters and hyperthyroid Graves' thyroids, PRDX5 immunoblotting was performed in these two respective tissues. We observed that PRDX5 expression was higher in the thyroid gland of patients with Graves' disease compared to multinodular goiters. In conclusion, our data show that PRDX5 is expressed in the thyroid gland where it could act as antioxidant. The level of expression is directly correlated with the functional status of epithelial cells, being higher in multinodular goiters, and even more pronounced in hyperthyroid tissues, such as Graves' disease.

Rat sciatic nerve regeneration within an acrylic semipermeable tube and comparison with a silicone impermeable material.

Sectioned rat sciatic nerves were placed in tubes to study the regrowth of injured nerve processes. First, we characterized quantitatively the regeneration of myelinated fibers at different levels of an acrylic semipermeable tube, from two to 27 weeks postoperatively. From the fifth week, myelinated fiber counts at mid-tube level were equal to the value of an intact nerve, but at the distal part the number of fibers exceeded both mid-tube level and unsectioned nerve values. At the proximal part of the tube an important fiber disorganization was observed. Second, we have compared acrylic semipermeable and totally impermeable silicone tubes at four and 27 weeks postoperatively. In terms of the number of myelinated fibers and the surface of the endoneurium at the mid-tube level, the most effective tube was the impermeable one. This study points out the importance of the tube wall permeability in nerve regeneration.

Comparison of the effects of auranofin and retinoic acid on plasminogen activator activity of peritoneal macrophages and Lewis lung carcinoma cells.

Urokinase-type plasminogen activator, a neutral proteinase, seems to play a central role in the degradation of the extracellular matrix that accompanies a number of biological phenomena including inflammatory reactions and neoplasia. The effect of auranofin and retinoic acid on the plasminogen activator activity expressed by two cell types, i.e. murine macrophages and Lewis lung carcinoma cells, has been investigated. Low concentrations of both drugs (10(-6)-10(-7) M) can inhibit in vitro the induction of plasminogen activator in macrophages stimulated by phorbol 12-myristate 13-acetate. This action occurs rapidly (15 min), is irreversible and is independent of a global cytotoxic effect. Auranofin and retinoic acid remain without effect in macrophages when added after stimulation by the phorbol ester. Both drugs are thus potent inhibitors of the induction of plasminogen activator activity in macrophages, possibly through an interaction with the protein kinase C system. The plasminogen activator activity of Lewis lung carcinoma cells, which is apparently not dependent on a protein kinase C pathway, is not influenced by auranofin or retinoic acid. These observations may contribute to explain: (1) the activity of auranofin and retinoic acid in rheumatoid arthritis, and (2) the antitumor promoting activity of retinoic acid. It would be relevant to assess whether auranofin may exhibit, like retinoic acid, an antitumor-promoting activity.

Plasminogen activator activity of normal and retinoic acid-treated post-implantation embryos.

Urokinase-type plasminogen activator (uPA) has been implicated in cellular migration accompanying different biological phenomena including organogenesis. An increase in uPA activity was observed in mouse post-implantation embryos during the early organogenesis period. Since we have previously shown that all-trans retinoic acid (RA) prevented the induction of uPA in mouse peritoneal macrophages, we have now assessed whether teratogenic doses of this agent could also interfere with uPA activity in mouse embryo in vitro and in vivo. Post-implantation embryos (8.5 days) were incubated for up to 24 hr with micromolar concentration of RA resulting in the occurrence of malformations. No significant difference in uPA activity was found between control and treated embryos. Likewise, uPA activity was not altered in embryos explanted on day 9.5 from dams treated 24 hr before with a teratogenic dose of RA. This study indicates that the teratogenic activity of RA is not caused by an inhibition of the induction of uPA in embryos.

Axonal regeneration after peripheral nerve grafting and fibrin-fibronectin-containing matrix implantation on the injured septohippocampal pathway of the adult rat: a light and electron microscopic study.

The damaged septohippocampal pathway was utilized to study the axonal regeneration of injured neurons. Semipermeable tubes, 2-mm long, were placed in the axis of the transected septohippocampal pathway of adult rats. In a first series of experiments, empty tubes were implanted. Even six weeks after the operation, no regenerated axons were observed in the conduit. In a second series of experiments, in order to validate our approach, segments of pre-degenerated sciatic nerves were introduced into the tubes. Under these experimental conditions, acetylcholinesterase (AChE)-containing regenerated axonal processes were detected in the grafted sciatic nerves. Glial fibrillary acidic protein (GFAP)-immunodetection showed that astroglial cells and astrocyte processes were able to progress on and into the peripheral grafts. At the electron microscopic level, axons were observed in close contact with Schwann cells which myelinated some of them. In some other cases, unmyelinated axons were also present at the surface of reactive astroglial cells filled by numerous intermediate filaments. These central glial cells had migrated among the sciatic nerve collagen fibers. No axon was detected without glial cell contact. In a third series of experiments, we implanted semipermeable tubes previously filled with a fibrin-fibronectin-containing matrix provided by peripheral regeneration chambers. One week after the implantation of the tubes containing this peripheral substrate, different cell types were observed migrating into the conduit and replacing the fibrin-fibronectin-containing matrix. Among these cells astrocytes were present as revealed by GFAP-immunocytochemistry and electron microscopic examinations. During the following weeks, axons were detected in contact with the reactive astroglial cells. AChE-histochemistry showed that axons were able to cross the two millimeter distance separating the septal part and the hippocampal part of the lesion site. GABA (γ-aminobutyric acid)-ergic fibers were also detected in the regenerated structure. These experiments show that cellular or acellular substrates provided by the PNS can promote the regeneration of CNS GABAergic and cholinergic neurons. Our observations suggest that astrocytes can take an important part, after their migration or after extending processes, in the axonal regeneration in the adult CNS of the rat, possibly in furnishing a cellular terrain for the progression of growth cones over a distance of two millimeters and in maintaining regenerated axons at least until the sixth week after the operation.

Clara cell secretory protein (CC16): features as a peripheral lung biomarker.

Clara cell protein (CC16 or CC10) is a 15.8-kDa protein secreted all along the tracheobronchial tree and especially in the terminal bronchioles where Clara cells are localized. Even though the exact in vivo function of CC16 remains to be clarified, evidence is accumulating that CC16 plays an important protective role in the respiratory tract against oxidative stress and inflammatory response. CC16, however, presents also a major interest as a peripheral lung marker for assessing the cellular integrity or the permeability of the lung epithelium. The serum concentrations of CC16 are decreased in subjects with chronic lung damage caused by tobacco smoke and other air pollutants as a consequence of the destruction of Clara cells. By contrast, serum CC16 increases in acute or chronic lung disorders characterized by an increased airways permeability. The sensitivity of serum CC16 to an increased leakiness of the lung allows for the detection of defects of the epithelial barrier at ozone levels below current air-quality guidelines. Although the clinical significance of these early epithelial changes detected by serum CC16 remains to be determined, these results clearly show that the assay in serum of lung secretory proteins such as CC16 represents a new noninvasive approach to evaluate the integrity of the respiratory tract.

Alpha 1-tubulin mRNA level is increased during neurite outgrowth of NG 108-15 cells but not during neurite outgrowth inhibition by CNS myelin.

alpha 1-tubulin is an isotype of alpha-tubulin, and its mRNA is expressed in the rodent nervous system. A high level of alpha 1-tubulin mRNA in neurones is associated with axonal outgrowth during development as well as with axonal regeneration after axotomy in adult animals. We quantitated alpha 1-tubulin mRNA levels in motor neurone-like NG 108-15 cells using Northern blots in order to determine whether the expression of this neurite outgrowth-associated gene is regulated in NG 108-15 cells during neurite extension and during inhibition of this process by CNS myelin. Here we report that during the acute phase of neurite outgrowth, alpha 1-tubulin mRNA level increases in NG 108-15, a maximal induction of 1.7-fold over the initial level occurring 24 h after neurite outgrowth onset. By contrast, when these cells are plated on CNS myelin alpha 1-tubulin mRNA levels show no such increase. These findings indicate that an increase of the alpha 1-tubulin mRNA level is associated with neurite outgrowth of NG 108-15 cells. More interestingly, this study also demonstrates that the inhibition of neurite outgrowth by CNS myelin may affect the expression of a gene encoding a protein involved in neurite extension.

Orientation of cell adhesion and growth on patterned heterogeneous polystyrene surface.

Studies of neurite outgrowth or cell migration, two important processes in neuronal networks formation, are facilitated by cell culture models capable of orientating cellular growth and of designing a well-defined cellular pattern. Heterogeneous polystyrene surfaces composed of oxygen plasma-treated stripes (PSox) with a low hydrophobicity separated by non-treated areas (PS) have these properties. In this study, to guide cell growth, we developed a cell culture model using these supports and we identified the molecular factors involved in cellular orientation. When the heterogeneous supports were not coated, proteins from a serum culture medium were required for cells to line up on PSox. On the other hand, cell orientation on coated surfaces was clearly influenced by competitive adsorption of adhesive proteins such as fibronectin or collagen and anti-adhesive molecules as pluronic F68 or albumin. Attachment factors were adsorbed on PSox stripes while adsorption of anti-adhesive molecules on the most hydrophobic PS areas prevented cell adhesion or growth. Thus, we describe the preparation of a cell culture substrate that succeeded in orientating cell growth and that led to a line of cells on adhesive PSox stripes ranging from 2 to 100 microns width.

Compartmentalized coculture of rat brain endothelial cells and astrocytes: a syngenic model to study the blood-brain barrier.

The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function.

A new in vivo model to study the influence of the microenvironment in the regeneration of the central nervous system.

In order to study the 'in vivo' regenerative capacity of the central nervous system, a semipermeable tube was placed in the axis of the lesioned nigrostriatal pathway of adult rats. In spite of a correct positioning of the tube, no growing central nervous processes were observed within the tube after 3 to 6 weeks when it was left empty. However, when the lumen of the tube was previously filled with a pre-degenerated sciatic nerve, unmyelinated and myelinated fibers were observed growing in the peripheral graft. Since the content of the tube can be modified, it appears that this model can be used to test the capability of cellular or acellular microenvironments to promote the 'in vivo' regeneration of the mammalian central nervous system fibers.

Axonal growth and glial migration from co-cultured hippocampal and septal slices into fibrin-fibronectin-containing matrix of peripheral regeneration chambers: a light and electron microscope study.

In order to investigate whether a fibrin-fibronectin-containing matrix of a peripheral regeneration chamber could promote the growth of central nervous system neurons, hippocampal and septal slices were co-cultured in the presence of this acellular substrate. In introducing the peripheral matrix into a 2-mm-long tube between hippocampal and septal slices, a spatio-temporal sequence of cell migration and axonal growth was described by light and electron microscopy. Axons were able to elongate directly into the flocculent material constituting the matrix and a possible neurite-promoting activity was implicated in this process as axonal growth was not detected in direct contact with rat plasma coagulated with calcium, or chicken plasma coagulated with thrombin, used as control matrices. However, in the 3 different substrates tested, astrocytes were able to migrate and dilated astroglial processes containing intermediate filaments were detected. Axonal processes were observed growing on the glial cell surface. GFAP-positive phagocytic cells, that could be of the same origin as astrocytes, were involved in matrix removing. Neuronal growth and glial migration arose from hippocampal and septum slices and acetylcholinesterase-containing fibers were seen in the bridging structure suggesting that cholinergic axons were able to progress to the hippocampal slice. This technique appeared to provide a model in which axonal growth and cell migration can be studied 'in vitro' in a 3-dimensional environment.

A new model for quantification of microvascular regeneration after a lesion of the rat cerebral cortex.

The purpose of this study is to validate a method for the quantification of the neovascularization in the vicinity of a lesion made in the cerebral rat cortex. A cavity, made by aspiration in the occipital cortex of young rats, induces around the lesion a parenchymal and vascular reaction. The parenchymal reaction is characterized by cellular necrosis and gliosis. The vascularization is more dense around the cavity than in normal cortex. Morphometric analysis indicates, 8 days after the lesion, a 130% increase of the total length of the vessels in comparison to the contralateral normal cortex.

Effects of dibutyryl cyclic adenosine monophosphate, ACTH(4-10) and gangliosides on nerve fiber regeneration after rat sciatic nerve tubulization.

Tubulization of sectioned rat sciatic nerves was used to evaluate the effect of dibutyryl cyclic adenosine monophosphate (db-cAMP), a fragment of the adrenocorticotrophic hormone (ACTH(4-10] and gangliosides on the regrowth of injured nerve processes. Four weeks post-operative, the number of myelinated fibers was counted at different levels of the lesioned sciatic nerves and the endoneurium surface of the bridging structure in the tube was measured. Animals treated with gangliosides and db-AMPc showed statistically significant differences compared to vehicle treated rats. The gangliosides have shown a beneficial effect but db-cAMP diminished the number of myelinated fibers. In this case, an implication of Schwann cell mitosis and migration is discussed. In spite of better results obtained in ACTH(4-10)-treated animals compared to controls, the differences did not attain a statistically significant level. These experiments confirm the beneficial effect of gangliosides on peripheral nerve regeneration and reveal a negative effect of db-cAMP in the regeneration chamber model.

Expression of integrins by murine MSC80 Schwann cell line: relationship to cell adhesion and migration.

Schwann cells (Sc) are one of the most important factors promoting regeneration of both the peripheral and the central nervous system. They provide a permissive environment for neurite outgrowth and the making of this environment requires interactions between Sc and extracellular matrix proteins that are mediated via integrin receptors. This study characterized, by immunoprecipitation, the integrins expressed by the mouse MSC80 Sc line. Our results showed that MSC80 Sc expressed alpha1beta1, alpha5beta1 and alpha6beta1 integrins as well as the alpha v-subunit associated with an unidentified 80-90 kDa beta-subunit. Adhesion and migration assays revealed a hierarchy of protein influences that are dependent upon the type of cellular behaviour. Integrin expression correlated with MSC80 Sc line adhesion and migration on extracellular matrix proteins. The MSC80 Sc line expressed a pattern of integrins which allowed adherence on vitronectin and collagen IV, and faster migration on merosin and laminin. As the integrin pattern and the behaviour of MSC80 on ECM were similar to primary Sc, MSC80 are a potential abundant source of Sc for further in vitro and in vivo experiments.

Regeneration of lesioned cholinergic septal neurons of the adult rat can be promoted by peripheral nerve grafts and a fibrin-fibronectin-containing matrix of peripheral regeneration chambers.

Axonal regeneration of septal cholinergic neurons was examined after lesion of the septohippocampal pathway of the adult rat and implantation of tubes containing peripheral cellular or acellular substrates. After empty tube implantation, no regenerated structures were observed in the conduit. However, after implanting tubes filled with sections of predegenerated sciatic nerves or a fibrin-fibronectin-containing matrix provided by peripheral regeneration chambers, numerous regenerated axons were detected 6 weeks after the operation. At the electron microscopic level, regenerated axons were observed in the grafted sciatic nerves in contact with Schwann cells but also in contact with astrocytes which were able to migrate and send processes into the graft. After fibrin-fibronectin-containing-matrix implantation, the regenerated structure between septum and hippocampus was composed mainly of fibroblasts, astrocytes, and regenerated axons associated to these central glial cells.

Suloctidil increases the rat brain cortex microvascular regeneration after a lesion.

A "cavity" lesion made by aspiration in the rat occipital cortex induces a parenchymal and a vascular reaction in its vicinity. The first was mainly characterized by cellular necrosis and gliosis, the second by an increase of the vascular network. In vehicle treated rats, a 50% significant increase of the vascular network was observed around the cavity 4 days after the lesion, in comparison to the uninjured contralateral cortex. The effects of a vasoactive substance, suloctidil, on the vascular reaction was studied in the brain cortex. A single oral dose of suloctidil (30 mg/kg; 2 hours before the sacrifice) gave the same effect as the vehicle group. After 8 days of suloctidil oral administration (30 mg/kg; twice daily: 4 days before lesion and 4 days after) a significant increase (123%) of the vascular network was observed around the cavity. The hypothetical ways by which a chronic treatment of suloctidil induces this increase of the neovascularization observed after cortical lesion are discussed.

Clara cell specific protein (CC16) expression after acute lung inflammation induced by intratracheal lipopolysaccharide administration.

Clara cell secretory protein (CC16, CC10, or CCSP), the major secretory protein of the Clara cell, presents several biologic properties, suggesting that it may play a protective role against intrapulmonary inflammatory processes. The aim of the present study was to investigate the changes of CC16 concentrations in the lung, bronchoalveolar lavage fluid (BALF), and serum of rats with acute lung injury induced by lipopolysaccharide (LPS). These changes were compared with Clara cell density, CC16 mRNA level in the lung and classic indices of inflammation in BALF. Injected at doses of 10, 100, or 200 microgram/100 g body weight, LPS induced an acute lung inflammation as estimated by an increased influx of cells and albumin in the BALF. This inflammatory response was associated with a marked reduction of CC16 concentrations in BALF and lung homogenate as well as of the CC16 mRNA levels in the lung. At the highest dose of LPS, the CC16-positive cell density in the bronchiolar epithelium was also decreased. In serum, by contrast, the concentration of CC16 was elevated as a consequence of increased airway permeability. Pretreating rats intraperitoneally with dexamethasone (2 mg/kg) significantly lowered the leukocyte influx and attenuated the albumin increase in BALF. Dexamethasone, however, failed to prevent the increased airway permeability to CC16, suggesting that during inflammation different mechanisms regulate the leakage of proteins across the alveolocapillary barrier depending on the direction of passage and/or the size of the protein. Our results show a marked decrease of the secretion and synthesis of CC16 during LPS-induced acute lung inflammation.

Clara cell protein as a marker of Clara cell damage and bronchoalveolar blood barrier permeability.

The 16 kDa Clara cell protein (CC16), an abundant component of airway secretions, has recently been proposed in humans as a pulmonary marker measurable not only in bronchoalveolar lavage fluid (BALF) but also in serum. The aim of the present study was to investigate the changes and determinants of CC16 concentrations in these fluids in normal rats and rats with lung injury. Female Sprague-Dawley rats were given a single i.p. injection of arachis oil (n=20) or chemicals in arachis oil (n=10) that mainly damage Clara cells (4-ipomeanol (IPO) 8 mg x kg(-1) and methylcyclopentadienyl manganese tricarbonyl (MMT) 5 mg x kg(-1)) or endothelial cells (alpha-naphthylthiourea (ANTU) 5 mg x kg(-1)). CC16 concentration (mean+/-sD in microg x L(-1)), measured by a sensitive latex immunoassay, was significantly reduced in BALF of all treated groups (IPO 380+/-100; MMT 730+/-200; ANTU 1,070+/-200; controls 1,700+/-470). The same pattern of decrease was observed in the labelling of Clara cells with an anti-CC16 antiserum as well as in the CC16 messenger ribonucleic acid levels assessed by Northern enzyme-linked immunosorbent assay. In serum, by contrast, CC16 was significantly increased in all treated groups (IPO 31+/-7; MMT 22+/-12; ANTU 52+/-24; controls 15+/-6). This rise of CC16 in serum was associated with an elevation of albumin in BALF which is an index of increased bronchoalveolar/blood barrier permeability. In conclusion, lung injury induces a decrease of the 16 kDa Clara cell protein in bronchoalveolar lavage fluid owing to a reduced production by damaged Clara cells, and an increase in serum protein levels resulting from its enhanced leakage across the bronchoalveolar/blood barrier. This study provides new insights into the understanding of the changes of lung secretory proteins in bronchoalveolar lavage fluid and serum.

Differential toxicity of ganciclovir for rat neurons and astrocytes in primary culture following adenovirus-mediated transfer of the HSVtk gene.

The toxicity of the suicide HSVtk gene approach is known to be targeted to DNA synthesis and, consequently, to dividing cells. This system is therefore useful for the treatment of brain tumors which contain dividing cells surrounded by a quiescent normal tissue. Adenoviruses are efficient vectors for the transfer of the HSVtk gene into the tumor but this can lead to the transduction of quiescent cells. In this study, we focused on the toxicity of the HSVtk/ganciclovir treatment for the two main cell types of the normal brain: astrocytes and neurons. Astrocytes and neurons in primary culture were infected by an adenoviral vector bearing the HSVtk gene (Ad.tk) and cells were exposed to different concentrations of ganclclovir. After 5 days of treatment, an MTT test measured a dramatic decrease in cell viability for treated astrocytes while a small decrease in cell viability was observed for neurons treated in the same experimental conditions. The differential toxicity of the HSVtk/ganciclovir treatment was also observed in cocultures of astrocytes and neurons: an immunocytochemical analysis of the treated cells showed major morphological modifications for astrocytes but not for neurons. Furthermore, our data suggest that a bystander effect is able to kill all the astrocytes while neurons from the same culture remain unaffected.