Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.

The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

Serum anticholinergic activity in patients following cardiac surgery and healthy individuals following amitriptyline application.

The serum anticholinergic activity (SAA) is used as a marker for cognitive impairment. Here, two studies have been performed characterizing the SAA profile. In Study 1 the endogenous SAA in relation to the total serum protein concentration was monitored for 24 h in five healthy individuals and compared with that in four inpatients following cardiac surgery. In Study 2 the SAA of seven healthy individuals was assessed following a single amitriptyline dose. In both studies SAA was assessed by an ex vivo assay. In Study 1, the absolute SAA varied in a wide range of 1.2 and 14.5 atropine equivalents (AEs) over 24 h. A circadian pattern was not observed. The mean total serum protein concentration, but not the SAA, was significantly lower in inpatients than in healthy individuals. In Study 2, the SAA increased following amitriptyline to a maximum. The mean SAA increased by 6.39 AE at the amitriptyline peak concentration. High SAA variability showed a low statistical relation to amitriptyline concentrations. Both studies characterize the SAA as an individual parameter not affected per se by surgery or clinical care and poorly correlated with the total serum protein concentration. The relation with amitriptyline concentration helps to quantify SAA values towards a better understanding of the clinical implications and limitations of SAA changes.

Induction of immediate early gene expression by high-frequency stimulation of the subthalamic nucleus in rats.

Deep brain stimulation is associated with delayed improvement of parkinsonian symptoms, such as hypokinesia with subthalamic nucleus stimulation, or dystonia with globus pallidus internus stimulation. The latency observed is better explained by molecular alterations than immediate electrophysiological processes, and clinical improvement may involve adaptive gene expression. Here, we have studied immediate early gene expression as fast molecular response to subthalamic nucleus stimulation. Bipolar electrodes were implanted bilaterally into the subthalamic nucleus of anesthetized male Wistar rats. High-frequency stimulation (130 Hz or 80 Hz, 60 micros, 300 microA) or low-frequency stimulation (5 Hz, 60 micros, 300 microA) was performed with the right electrode for 15, 60, 120, and 240 min whereas the silent left electrode served as negative control. Brains were fixed by transcardial perfusion and frozen sections were stained with polyclonal antibodies directed against three immediate early gene-encoded proteins, c-Fos, c-Jun, and Krox-24 (NGFI-A, Egr-1, Zif268, Tis8, Zenk). After 120 and 240 h, c-Fos immunoreactivity was strongly upregulated in subthalamic nucleus neurons on the stimulated site. In contrast, no c-Fos immunoreactivity was detected on the non-stimulated site except for single positive cells located in close proximity to the electrode tracks. Furthermore, c-Fos immunoreactivity was induced in subthalamic nucleus projection areas, such as primary and secondary motor cortex, primary somatosensory and insular cortex, lateral and medial globus pallidus, suprageniculate thalamic nucleus, pontine nuclei, medial geniculate nucleus, and substantia nigra. Similarly, c-Jun and Krox-24 were induced at the site of stimulation and in projection areas following high-frequency subthalamic nucleus stimulation. Whereas high frequency stimulation with 80 Hz was similarly effective none of the three immediate early gene-encoded proteins was induced with low-frequency stimulation (5 Hz) for 4 h. This is in accordance with the therapeutic effects of deep brain stimulation which are only elicited with high frequency stimulation. Our data provide evidence that immediate early gene expression in the subthalamic nucleus is rapidly and substantially induced by high-frequency stimulation. The induction of immediate early genes in projection sites suggests ipsilateral transsynaptic modulation of neuronal activity.

Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii.

We have developed a control system for regulating gene activation in Toxoplasma gondii. The elements of this system are derived from the Escherichia coli tetracycline resistance operon, which has been widely used to tightly control gene expression in eukaryotes. The tetracycline repressor (tetR) interferes with transcription initiation while the chimeric transactivator, composed of the tetR fused to the activating domain of VP16 transcriptional factor, allows tet-dependent transcription. Accordingly, tetracycline derivatives such as anhydrotetracycline, which we found to be well tolerated by T.gondii, can serve as effector molecules, allowing control of gene expression in a reversible manner. As a prerequisite to functionally express the tetR in T.gondii, we used a synthetic gene with change of codon frequency. Whereas no activation of transcription was achieved using the synthetic tetracycline-controlled transactivator, tTA2(s), the TetR(s )modulates parasite transcription over a range of approximately 15-fold as measured for several reporter genes. We show here that the tetR-dependent induction of the T.gondii myosin A transgene expression drastically down-regulates the level of endogenous MyoA. This myosin is under the control of a tight feedback mechanism, which occurs at the protein level.

Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre.

Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system.

Activity and expression of JNK1, p38 and ERK kinases, c-Jun N-terminal phosphorylation, and c-jun promoter binding in the adult rat brain following kainate-induced seizures.

The activity and/or expression of the mitogen-activated protein kinases c-Jun N-terminal kinase 1, p38 and extracellular signal-regulated kinases 1/2, as well as their substrates, the transcription factors c-Jun and activating transcription factor-2, were examined following systemic application of kainate in the cortex and hippocampus of the adult rat brain. The protein expression levels of all three mitogen-activated protein kinases remained constant during the observation period. Unexpectedly, c-Jun N-terminal kinase 1 was the only mitogen-activated protein kinase activated in this model of excitotoxicity, its activity raised from between 1 and 3 h moderate basal to maximal levels between 6 and 12 h. In contradistinction, activity of extracellular signal-regulated kinases 1/2 fell from their substantial basal levels and did not recover; activity of p38 was characterized by a high basal level that almost entirely disappeared and did not return to basal levels even 10 days after kainate application. c-Jun protein was rapidly expressed, with a maximum after 3 h and a slow decline after 12 h. Supershift assays revealed that, during the early induction phase of the c-jun gene, the proximal activator protein-1 (jun1) site of the c-jun promoter was mainly occupied by the constitutively expressed activating transcription factor-2, whereas the late induction correlated with the predominant binding of c-Jun and, to a lesser extent, activating transcription factor-2 to the distal activator protein-1 (jun2) site. The time-course of the N-terminal phosphorylation of c-Jun as determined by immunocytochemistry paralleled the activity of c-Jun N-terminal kinase 1 and showed a compartment-specific regulation between 3 and 12 h. A second set of supershift experiments demonstrated that c-Jun, but not activating transcription factor 2, bound to activator protein-1 sites in the promoter of substance P and collagenase genes, but not of the cyclo-oxygenase-2 gene. Our results demonstrate that activation of c-Jun N-terminal kinase 1, phosphorylation of c-Jun and selective occupation of the c-jun promoter by activating transcription factor-2 or c-Jun are part of the neuronal response following excitotoxicity that is considered as the mechanism for neuronal apoptosis in vivo. Some of these findings differ substantially from in vitro experiments and underline the necessity to analyse the neuronal stress pathways in the adult brain.

Changes in peptidyl-prolyl cis/trans isomerase activity and FK506 binding protein expression following neuroprotection by FK506 in the ischemic rat brain.

FK506 is an immunosuppressant also showing neuroprotection following cerebral ischemia. FK506 binds to intracellular proteins (FKBP) which have a wide range of functions but have in common the peptidyl-prolyl cis/trans isomerase activity. Following transient focal ischemia, we have analyzed the expression of FKBP12, 52 and 65 and the total FKBP enzyme activity. Furthermore, we have investigated the effect of FK506 on signal transduction in neurons and perfusion changes in the infarct area. After 90 min of transient middle cerebral artery occlusion in male rats the expression of FKBP12, 52 and 65 was analyzed by Western blot in FK506-treated and control animals and the peptidyl-prolyl cis/trans isomerase activity was determined. Magnetic resonance imaging was used to measure tissue perfusion, development of vasogenic edema and infarct size. To investigate the neuronal stress signal cascade, activating transcription factor 2 (ATF-2), Fas-ligand (Fas-L) and c-Jun expression and phosphorylation were analyzed by immunohistochemistry. FK506 decreased the cerebral infarct volume by 53% and reduced the cytotoxic edema. The total FKBP enzymatic activity in the infarct area was increased and blocked dose dependently by FK506. FKBP expression was selectively up-regulated by cerebral ischemia. FK506 treatment does not influence the expression patterns. c-Jun phosphorylation in neurons of the peri-infarct area and Fas-L expression was reduced by FK506 treatment whereas ATF-2 expression was preserved. Cerebral ischemic damage to the brain was reduced by FK506. It was shown for the first time that neuroprotection by FK506 also included the suppression of the cerebral peptidyl-prolyl cis/trans isomerase activity of FKBP in vivo whereas the expression levels of FKBP12, 52 and 65 following ischemia changed slightly and FK506 treatment does not suppress the expression patterns. However, changes of FKBP enzymatic activity result in suppression of the stress cell body response in the peri-infarct area as observed by suppression of c-Jun phosphorylation and Fas-L expression.

The microneme protein MIC4, or an MIC4-like protein, is expressed within the macrogamete and associated with oocyst wall formation in Toxoplasma gondii.

The expression and localisation of MIC4, or an immuno-cross reacting MIC4-like protein, was examined in the enteric forms of Toxoplasma gondii using immunocytochemistry. In addition to being located within the micronemes of the merozoites, MIC4 or the MIC4-like protein was present within the macrogamete and was associated with the developing oocyst wall. The macrogamete is characterised by two types of structurally distinct wall forming bodies (WFB1 and 2). However, by immuno-electron microscopy, it was possible to identify two populations of dense granules (WFB1) which appear to form sequentially during macrogamete development. The first granules to form (WFB1a) stained positively with anti-MIC4 and were followed by MIC4 negative granules (WFB1b). During oocyst wall formation, the WFB1a and b sequentially released their contents onto the surface with WFB1a material forming an anti-MIC4 positive outer veil, while the WFB1b forms the electron dense outer layer of the oocyst wall. The inner layer was formed by WFB2. Thus, for the first time, it was possible to identify two populations of dense granules (WFB1a and b) involved in the formation of different parts of the oocyst wall. It was not possible to analyse the contents of macrogametes by western blot to unequivocally identify the antigen recognised by the polyclonal antisera as MIC4.

The transcription factor CREB is not phosphorylated at serine 133 in axotomized neurons: implications for the expression of AP-1 proteins.

The present study has investigated whether nerve fiber transection alters the phosphorylation of serine at position 133 (Ser133) of the transcription factor CREB (phosphoCREB). Activation of CREB by phosphorylation has a major function in the control of gene transcription. PhosphoCREB was visualized by antisera that specifically react with an epitope comprising the phosphorylated Ser133 of CREB as well as of CREM and ATF1 proteins. In untreated rats, nuclear immunoreactivity (IR) of phosphoCREB was consistently visible, e.g. in the cortex, thalamic and hypothalamic compartments and central termination areas of primary somatosensory afferents. Transection of peripheral (sciatic nerve), cranial (hypoglossal and facial nerve) and central (medial forebrain bundle and mammillo-thalamic tract) nerve fibers did not increase phosphoCREB-IR in the axotomized neurons between 5 min and 30 days post-axotomy. In contrast, phosphoCREB-IR appeared after 24 h in glial cells adjacent to the axotomized motoneurons and persisted up to 4 weeks. This increase in glial phosphoCREB-IR was paralleled by enhanced expression of the CREB protein itself. Between 20 min and 24 h following sciatic nerve transection, the number of phosphoCREB labeled nuclei also increased in neurons of the ipsilateral superficial dorsal horn of lumbar L3-L5 spinal cord segments. These data suggest that phosphorylation of Ser133 in CREB/CREM/ATF1 proteins is not involved in the transcriptional control of early-response genes such as c-jun in axotomized neurons following nerve transection. This is in contrast to the reported phosphorylation of CREB and its trans-acting effects on immediate-early genes such as c-fos after transynaptic neuronal excitation.

Persisting expression of galanin in axotomized mamillary and septal neurons of adult rats labeled for c-Jun and NADPH-diaphorase.

In adult male rats, the expression of the neuropeptide galanin and its co-localization with the c-Jun transcription factor and the NADPH-diaphorase, the marker enzyme for the nitric oxide synthase (NOS), was investigated by immunohistochemistry in axotomized neurons following unilateral stereotaxic transection of the (a) mamillo-thalamic tract, (b) medial forebrain bundle, (c) fimbria fornix bundle and (d) sciatic nerve. This surgical procedure resulted in axotomy of neurons of (a) mamillary ncl. (MnM), (b) substantia nigra compacta (SNC) and paraventricular ncl. of thalamic (PF) neurons, (c) medial septum (MS) and vertical diagonal band of Broca (VDB), and (d) sciatic motoneurons and dorsal root ganglia (DRG). In all of these axotomized neuronal subpopulations, expression of c-Jun appeared between 24 and 36 h post-axotomy and persisted on substantial levels for 15 days in the SNC and for 30-50 days in the MnM, PF, MS, VBD, sciatic DRG and motoneurons. Expression of galanin was seen in axotomized MnM, MS and DRG, but not in SNC, PF and sciatic motoneurons. Galanin-immunoreactivity (IR) appeared between 3 and 5 days after nerve fiber transection and persisted up to 50 days in the MnM, MS and DRGs. The cytoplasmic galanin-IR was almost completely restricted to those neurons showing a nuclear c-Jun expression. Moreover, galanin expression showed a long-lasting co-localization with those neurons that exhibited an increased NADPH-diaphorase reactivity in the MnM and DRG or a residual NADPH-diaphorase reactivity in MS post-axotomy. Very similar to galanin, NADPH-diaphorase was not affected by axotomy in the SNC, PF or sciatic motoneurons. Our findings suggest a common mechanism for galanin and NOS (NADPH-diaphorase activity) expression. Since the galanin promotor contains an AP-1 binding site, c-Jun might trigger the lasting induction of galanin in NOS-positive central neurons that survive the axotomy-evoked injury.

Rapid and long-lasting suppression of the ATF-2 transcription factor is a common response to neuronal injury.

The activating transcription factor 2 (ATF-2) protein, a neuronal constitutively expressed CRE-binding transcription factor, is essential for the intact development of the mammalian brain. ATF-2 is activated by c-Jun N-terminal kinases and modulates both the induction of the c-jun gene and the function of the c-Jun protein, a mediator of neuronal death and survival. Here we show by immunocytochemistry and Western blotting that ATF-2 is rapidly suppressed in neurons within 1-4 h following neuronal stress such as transient focal ischemia by occlusion of the medial cerebral artery, mechanical injury of the neuroparenchym, stimulation of adult dorsal root ganglion neurons in vitro by doxorubicin as well as within 24 h following nerve fiber transection. ATF-2 reappears and regains basal levels between 12 h and 72 h following ischemia, between 50 and 100 days following axotomy, but remains absent around the site of mechanical injury during the process of degeneration. Following ischemia and tissue injury, ATF-2-IR also disappeared in areas remote from the affected brain compartments indicating the regulation of its expression by diffusible molecules. These findings demonstrate that the rapid and persistent down-regulation of ATF-2 is a constituent of the long-term neuronal stress response and that the reappearance of ATF-2 after weeks is a marker for the normalization of neuronal gene transcription following brain injury.

Repetitive electroconvulsive seizures induce activity of c-Jun N-terminal kinase and compartment-specific desensitization of c-Jun phosphorylation in the rat brain.

Electroconvulsive seizures (ECS) are used for therapy of pharmacoresistent depression and are supposed to induce long-lasting neuronal alterations in morphology and gene expression. In this study, we have investigated the phosphorylation of the transcription factor protein c-Jun at its serine 73 residue by immunohistochemistry and the activity of the c-Jun N-terminal kinase 1 (JNK1) by immunocomplex assay following repetitive ECS in adult rats. In untreated controls, nuclear c-Jun immunoreactivity, but not N-terminal phosphorylation, was present in a variety of neuronal populations including the hippocampus, the temporobasal cortex and the amygdalar complex. Daily ECS for 1, 5 or 10 days (1x, 5x or 10x ECS) did not alter the expression of c-Jun but caused a substantial N-terminal phosphorylation of c-Jun (phospho-c-Jun). Nuclear phospho-c-Jun immunoreactivity was maximal within 15 min following ECS, and became absent after 30 min. The highest levels of phospho-c-Jun labeling were found after 1x ECS in the amygdalar complex, the dorsomedial hypothalamus and the piriform cortex. The inducibility of c-Jun N-terminal phosphorylation was preserved in the medial amygdala and piriform cortex, but significantly declined in the basal amygdala and medial hypothalamus with progressive ECS stimulation. One single ECS 3 or 5 days following 10x ECS yielded a pattern of phospho-c-Jun as seen following 10x ECS; thus, a lag of 5 days was not sufficient to provoke the initial level of N-terminal phosphorylation of c-Jun. In the rostral hippocampus, c-Jun was not phosphorylated at any investigated time inspite of its high constitutive expression. In some contrast with this compartment-specific phosphorylation of c-Jun, immunocomplex assays revealed that the JNK1 activity was strongly enhanced in both amygdala and hippocampus. Our findings demonstrate that rapid JNK activation and phosphorylation of c-Jun as stand-by transcription factor characterize the beginning of neuroplastic changes, e.g., following ECS, a classic treatment of mental disorders. The N-terminal phosphorylation is compartment specific and can habituate following repetitive stimulation suggesting that the differential activation of the JNK/c-Jun axis is part of the neuronal strategy to integrate transynaptic excitation.

Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity.

Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Contributing mechanisms and markers of cell death are the activation of caspase-3 and DNA fragmentation. It remains to be resolved to which extent both cellular reactions overlap and/or indicate different processes of neurodegeneration. In this study, mixed neuronal cultures from newborn mice pubs (0-24 h) were stimulated with glutamate, and the co-localization of active caspase-3 and DNA fragmentation was investigated by immunocytochemistry and the TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (marked by MAP-2) displayed activated caspase-3 at different morphological stages of degeneration. TUNEL staining was detected in 5% of cell nuclei including GFAP-positive astrocytes. However, co-localization of active caspase-3 with TUNEL was less than 2%. After glutamate stimulation (125 microM), the majority of neurons was dying between 12 and 24 h. The absolute number of active caspase-3 neurons increased only moderately but in relation of surviving neurons after 24 h from 8 to 36% (125 microM), to 53% (250 microM) or to 32% (500 microM). TUNEL staining also increased after 24 h following glutamate treatment to 37% but the co-localization with active caspase-3 remained at the basal low level of 2%. In our system, glutamate-mediated excitotoxicity effects the DNA fragmentation and caspase-3 activation. Co-localization of both parameters, however, is very poor. Active caspase-3 in the absence of TUNEL indicates a dynamic degenerative process, whereas TUNEL marks the end stage of severe irreversible cell damage regardless to the origin of the cell.

[Enflurane anaesthesia for epileptic patients (author's transl)]. / Enfluran-Anaesthesien bei Epileptikern.

Enflurane anaesthesia with continuous EEG-registration was given to 21 patients, aged 3 to 53 years, who had a history of cerebral convulsions. 11 patients were children up to the age of 14 years. There was no obvious changes or correlation between enflurane concentration and intraoperative EEG-findings. The registered cramp potentials were the same as those registered preceeding the operations. Provocation as a result of the enflurane anaesthesia was not observed in any patient. The anticonvulsive basic treatment preceeding the operations made the interpretation of our results difficult. The warning by some authors against the use of Enflurane anaesthesia in patients with a history of cerebral convulsions could not be confirmed. Therefore, we conclude that a history of cerebral convulsions is not a general contraindication for the use of enflurane.

Isolation and characterization of genetically engineered gallidermin and epidermin analogs.

Gallidermin (Gdm) and epidermin (Epi) are highly homologous tetracyclic polypeptide antibiotics that are ribosomally synthesized by a Staphylococcus gallinarum strain and a Staphylococcus epidermidis strain, respectively. These antibiotics are secreted into media and are distinguished by the presence of the unusual amino acids lanthionine, 3-methyllanthionine, didehydrobutyrine, and S-(2-aminovinyl)-D-cysteine, which are formed by posttranslational modification. To study the substrate specificities of the modifying enzymes and to obtain variants that exhibit altered or new biological activities, we changed certain amino acids by performing site-specific mutagenesis with the Gdm and Epi structural genes (gdmA and epiA, respectively). S. epidermidis Tü3298/EMS6, an epiA mutant of the Epi-producing strain, was used as the expression host. This mutant synthesized Epi, Gdm, or analogs of these antibiotics when the appropriate genes were introduced on a plasmid. No Epi or Gdm analogs were isolated from the supernatant when (i) hydroxyamino acids involved in thioether amino acid formation were replaced by nonhydroxyamino acids (S3N and S19A); (ii) C residues involved in thioether bridging were deleted (delta C21, C22 and delta C22); or (iii) a ring amino acid was replaced by an amino acid having a completely different character (G10E and Y20G). A strong decrease in production was observed when S residues involved in thioether amino acid formation were replaced by T residues (S16T and S19T). A number of conservative changes at positions 6, 12, and 14 on the Gdm backbone were tolerated and led to analogs that had altered biological properties, such as enhanced antimicrobial activity (L6V) or a remarkable resistance to proteolytic degradation (A12L and Dhb14P). The T14S substitution led to simultaneous production of two Gdm species formed by incomplete posttranslational modification (dehydration) of the S-14 residue. The fully modified Dhb14Dha analog exhibited antimicrobial activity similar to that of Gdm, whereas the Dhb14S analog was less active. Both peptides were more sensitive to tryptic cleavage than Gdm was.

Long-lasting expression of JUN and KROX transcription factors and nitric oxide synthase in intrinsic neurons of the rat brain following axotomy.

In adult rats, the medial forebrain bundle (MFB) and mammillothalamic tract (MT) were unilaterally transected, resulting in axotomy of neurons in numerous areas such as the substantia nigra (SN), ventral tegmental area (VTA), nucleus (ncl.) mammillaris (MnM), and ncl. parafascicularis of the thalamus (PF). In these areas, expression of the transcription factor proteins c-JUN, JUN B, JUN D, c-FOS, FOS B, KROX-20, KROX-24, and CREB was investigated by immunocytochemistry up to 150 d. In parallel, the expression of nitric oxide synthase (NOS) was investigated both immunocytochemically and by the NADPH-diaphorase reaction (NDP), and the antibody against NOS was further characterized. The colocalization of c-JUN with NDP or NOS was also studied in the axotomized neurons. c-JUN and JUN D became visible in nuclei of many neurons of the ipsilateral MnM, PF, VTA, and SN (predominantly in the pars compacta and those double labeled by tyrosine hydroxylase, TH) after 36 hr, not after 24 hr, following transection of MFB and MT. In MnM, c-JUN and JUN D persisted at a nearly maximal level for up to 150 d. In PF, these proteins returned to control levels after 75 d. Expression of c-JUN and JUN D declined in the VTA after 30 d, but in the SN, it already declined after only 10 d. KROX-24 had a later onset of expression, being visible after 3 d in all investigated areas, and its pattern was similar to that of JUN proteins, although labeling was visible in fewer nuclei and declined earlier. JUN B, c-FOS, FOS B, and KROX-20 were not expressed in these areas, and substantial alterations of CREB immunoreactivity (CREB-IR) could not be detected. A subset of SN neurons (predominantly in the pars reticularis and negative for TH) presented an early and transient expression of all studied JUN, FOS, and KROX-24 proteins within 3 hr of transection that declined between 24 hr and 48 hr to basal levels. This expression pattern is typical of that caused by transynaptic stimulation (probably due to excitation of descending striatal neurons running within the MFB) and was clearly distinct from that evoked by c-JUN, JUN D, and KROX-24 IRs after 36 hr (predominantly in the pars compacta). An ipsilateral increase in NOS and NDP became visible in many neurons of the MnM after 10 d, but not after 5 d, and this persisted up to 150 d. The temporospatial pattern of NDP was similar to the pattern of NOS-IR.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of c-Jun and suppression of CREB transcription factor proteins in axotomized neurons of substantia nigra and covariation with tyrosine hydroxylase.

In adult rats, the expression of transcription factor proteins c-Jun and CREB and their colocalization with tyrosine hydroxylase (TH) were investigated in neurons of the substantia nigra compacta (SNC) axotomized by stereotaxic unilateral transection of the medial forebrain bundle (MFB). Axotomized SNC neurons were identified by injection of the retrograde tracer horseradish-peroxidase-coupled-gold (HRP-gold) into the ipsilateral striatum 5 days prior to MFB transection. Nuclear c-Jun immunoreactivity (IR) appeared 36 h after MFB transection in SNC neurons, was maximal after 5 days, and declined after 10 days. c-Jun-IR was visible in HRP-gold-labeled SNC neurons, demonstrating that c-Jun is in fact expressed in axotomized neurons. The constitutively expressed CREB (calcium/cAMP response element-binding protein, syn. CREB-1) was present in apparently all neuronal and glial cells in the brains of untreated rats including those SNC neurons that coexpressed TH. Three days following MFB transection, the nuclear CREB-IR disappeared in the axotomized SNC neurons labeled by TH-IR and was almost completely absent after 20 days in this neuronal population. The TH-IR rapidly declined 5 days after MFB transection, and 10 and 100 days post-axotomy the number of TH-labeled neurons was reduced by 52 and 80%, respectively. During this period, the majority of surviving TH positive neurons coexpressed c-Jun but were immunonegative for CREB. Between 3 and 60 days following MFB transection, the number of CREB-labeled glial cell nuclei increased in the ipsilateral substantia nigra by about 80%. Concomitantly, expression of GFAP, a marker protein for astrocytes, was also enhanced whereas nuclear c-Jun-, JunD-, and c-Fos-IR did not change in glial cells. These findings demonstrate that c-Jun can be expressed in axotomized neurons during the absence of CREB and suggest a role of c-Jun in the transcriptional control of the TH gene.

Transection of rat fimbria-fornix induces lasting expression of c-Jun protein in axotomized septal neurons immunonegative for choline acetyltransferase and nitric oxide synthase.

The fimbria-fornix (FF) fiber tract was unilaterally transected in adult rats by a stereotaxic knife cut. In the axotomized neurons of the medial septal nucleus (MS) and ventral diagonal band of Broca (VDB), the expression of Jun, Fos, Krox, CREB transcription factors, choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) were studied by immunocytochemistry. In addition, NADPH-diaphorase (NDP) and acetylcholine esterase (AChE) were visualized by activity assays. For retrograde tracing of axotomized neurons, either HRP-coupled gold was injected in the entorhinal cortex prior to axotomy, or Fast Blue was injected into the transection site subsequently to FF transection. Following FF transection c-Jun and in a less extend JunD were expressed in axotomized MS and VDB neurons. Expression levels rose at 24 h, but not at 18 h, postaxotomy, reached their maximal levels between 5 and 7 days, and then gradually declined. Up to 100 days, c-Jun was still present in a substantial number of septal neurons. JunB, Krox-20, Krox-24, c-Fos, and pan-Fos immunoreactivities (IR) were not detectable in axotomized septal neurons and CREB-IR did not change compared to the intact contralateral side. ChAT-IR dramatically declined over 36 h, and furthermore AChE reactivity had substantially fallen after 5 days. The number and intensity of cytoplasmic neuronal NOS-IR and NDP which generated congruent temporospatial patterns gradually fell between 3 and 5 days postaxotomy. The surviving neurons labeled by NOS and NDP showed a high coexpression of c-Jun, whereas c-Jun was almost completely absent in neurons stained for ChAT and AChE. Finally, ChAT-IR and NDP reaction labeled different subpopulations. Our findings demonstrate a lasting expression of the c-Jun transcription factor in axotomized MS and VDB neurons that might indicate the regenerative propensity of damaged neurons. The decrease of NOS and NDP in MS and VDB neurons demonstrates that neuronal populations respond to axotomy with an individual regulation of NOS expression.

The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains.

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.