Structural studies of calcium silicate hydrate modified with heavy metal cations.

This study investigates the immobilization mechanisms of heavy metal ions in the C-S-H phase. Synthetic C-S-H phases were prepared via the precipitation method, incorporating five different ions (Pb(II), Cd(II), Ni(II), Zn(II), and Cr(III)). Structural analysis of the obtained material was conducted using vibrational spectroscopy (both FT-IR and Raman), X-ray photoelectron spectroscopy, and X-ray diffraction. Spectroscopic methods were primarily employed to evaluate the structural effects and polymerization degree of the resulting C-S-H phase. Morphological changes were characterized using scanning and transmission electron microscopy (SEM and TEM, respectively). Our findings reveal several mechanisms for immobilizing heavy metal cations: precipitation of insoluble compounds (particularly notable for Ni(II) and Cr(III)), replacement of Ca(II) ions within the silicate structure (evident in the crystallization of Ca(OH)2 in samples containing Cd(II), Ni(II), and Zn(II) in minimal quantities), and strong bonding of certain metals (such as Pb(II)) with the C-S-H phase structure. These insights contribute to understanding the potential applications of C-S-H phases in heavy metal immobilization.

Living on the edge: the role of Atgolgin-84A at the plant ER-Golgi interface.

The plant Golgi apparatus is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Golgi matrix components, such as golgins, have been identified and suggested to function as putative tethering factors to mediate the physical connections between Golgi bodies and the ER network. Golgins are proteins anchored to the Golgi membrane by the C-terminus either through transmembrane domains or interaction with small regulatory GTPases. The golgin N-terminus contains long coiled-coil domains, which consist of a number of α-helices wrapped around each other to form a structure similar to a rope being made from several strands, reaching into the cytoplasm. In animal cells, golgins are also implicated in specific recognition of cargo at the Golgi.Here, we investigate the plant golgin Atgolgin-84A for its subcellular localization and potential role as a tethering factor at the ER-Golgi interface. For this, fluorescent fusions of Atgolgin-84A and an Atgolgin-84A truncation lacking the coiled-coil domains (Atgolgin-84AΔ1-557) were transiently expressed in tobacco leaf epidermal cells and imaged using high-resolution confocal microscopy. We show that Atgolgin-84A localizes to a pre-cis-Golgi compartment that is also labelled by one of the COPII proteins as well as by the tether protein AtCASP. Upon overexpression of Atgolgin-84A or its deletion mutant, transport between the ER and Golgi bodies is impaired and cargo proteins are redirected to the vacuole. LAY DESCRIPTION: The Golgi apparatus is a specialised compartment found in mammalian and plant cells. It is the post office of the cell and packages proteins into small membrane boxes for transport to their destination in the cell. The plant Golgi apparatus consist of many separate Golgi bodies and is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Specialised proteins called golgins have been suggested to tether Golgi bodies and the ER. Here we investigate the plant golgin Atgolgin-84A for its exact within the Golgi body and its potential role as a tethering factor at the ER-Golgi interface. For this, we have fused Atgolgin-84A with a fluorescent protein from jellyfish and we are producing this combination in tobacco leaf cells. This allows us to see the protein using laser microscopy. We show that Atgolgin-84A localises to a compartment between the ER and Golgi that is also labelled by the tether protein AtCASP. When Atgolgin-84A is produced in high amounts in the cell, transport between the ER and Golgi bodies is inhibited and proteins are redirected to the vacuole.

Human Wharton's jelly mesenchymal stem cells: properties, isolation and clinical applications.

Human Wharton's jelly mesenchymal stem cells (WJ-MSCs) exhibit CD29, CD79 and CD105 markers, characteristic for mesenchymal cell lines. Under the influence of the appropriate factors, WJ-MSCs can be dedifferentiated to osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes, glial cells and dopaminergic neurons. Wharton's jelly (WJ) is one of the potential sources of mesenchymal stem cells (MSCs) - obtaining these cells does not raise moral or ethical objections, because the umbilical cord (UC) is a regular waste material. The expression of the OCT-4 and Nanog proteins, which are characteristic for WJ-MSCs may indicate that these cells have retained some embryonic character. The collected data suggests that WJMSCs show increased division and telomerase activity compared to bone marrow MSCs (BM-MSCs). The published results showed no human leucocyte antigen (HLA) class II expression, with the possibility of HLA class I modification by WJ-MSCs, allowing for the transplantation of these cells both within the same and other species - which allows the use of human cells in animal models. The results of selected studies indicate that WJ-MSCs can be an essential element of regenerative medicine of the 21st century.

Amyloid precursor protein, lipofuscin accumulation and expression of autophagy markers in aged bovine brain.

BACKGROUND: Autophagy is a highly regulated process involving the bulk degradation of cytoplasmic macromolecules and organelles in mammalian cells via the lysosomal system. Dysregulation of autophagy is implicated in the pathogenesis of many neurodegenerative diseases and integrity of the autophagosomal - lysosomal network appears to be critical in the progression of aging. Our aim was to survey the expression of autophagy markers and Amyloid precursor protein (APP) in aged bovine brains. For our study, we collected samples from the brain of old (aged 11-20 years) and young (aged 1-5 years) Podolic dairy cows. Formalin-fixed and paraffin embedded sections were stained with routine and special staining techniques. Primary antibodies for APP and autophagy markers such as Beclin-1 and LC3 were used to perform immunofluorescence and Western blot analysis. RESULTS: Histologically, the most consistent morphological finding was the age-related accumulation of intraneuronal lipofuscin. Furthermore, in aged bovine brains, immunofluorescence detected a strongly positive immunoreaction to APP and LC3. Beclin-1 immunoreaction was weak or absent. In young controls, the immunoreaction for Beclin-1 and LC3 was mild while the immunoreaction for APP was absent. Western blot analysis confirmed an increased APP expression and LC3-II/LC3-I ratio and a decreased expression of Beclin-1 in aged cows. CONCLUSIONS: These data suggest that, in aged bovine, autophagy is significantly impaired if compared to young animals and they confirm that intraneuronal APP deposition increases with age.

Functional characterization of the ectopically expressed olfactory receptor 2AT4 in human myelogenous leukemia.

The olfactory receptor (OR) family was found to be expressed mainly in the nasal epithelium. In the last two decades members of the OR family were detected to be functional expressed in different parts of the human body such as in liver, prostate or intestine cancer cells. Here, we detected the expression of several ORs in the human chronic myelogenous leukemia (CML) cell line K562 and in white blood cells of clinically diagnosed acute myeloid leukemia (AML) patients by RT-PCR and next-generation sequencing. With calcium-imaging, we characterized in greater detail the cell biological role of one OR (OR2AT4) in leukemia. In both cell systems, the OR2AT4 agonist Sandalore-evoked strong Ca(2+) influx via the adenylate cyclase-cAMP-mediated pathway. The OR2AT4 antagonist Phenirat prevented the Sandalore-induced intracellular Ca(2+) increase. Western blot and flow cytometric experiments revealed that stimulation of OR2AT4 reduced the proliferation by decreasing p38-MAPK phosphorylation and induced apoptosis via phosphorylation of p44/42-MAPK. Furthermore, Sandalore increased the number of hemoglobin-containing cells in culture. We described for the first time an OR-mediated pathway in CML and AML that can regulate proliferation, apoptosis and differentiation after activation. This mechanism offers novel therapeutic options for the treatment of AML.

Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells.

The ectopic expression of olfactory receptors (ORs) in the human body has been of major interest in the past decade. Several studies have reported the expression of ORs not only in healthy tissues such as heart, sperm or skin cells, but also in cancerous tissues of the liver, prostate or intestine. In the present study, we detected the expression of OR51B5 in the chronic myelogenous leukemia (CML) cell line K562 and in white blood cell samples of clinically diagnosed acute myelogenous leukemia (AML) patients by reverse transcription-PCR and immunocytochemical staining. The known OR51B5 ligand isononyl alcohol increased the levels of intracellular Ca(2+) in both AML patient blood cells and K562 cells. With calcium imaging experiments, we characterized in greater detail the OR51B5-mediated signaling pathway. Here, we observed an involvement of adenylate cyclase and the downstream L-type and T-type calcium channels. In addition, the activation of OR51B5 leads to an inhibition of cell proliferation in K562 cells. In western blot experiments, we found that incubation with isononyl alcohol led to a reduction in p38-MAPK (mitogen-activated protein kinase) phosphorylation that might be responsible for the decreased cell proliferation. In the present study, we characterized the OR51B5-mediated signaling pathway downstream of the activation with isononyl alcohol, which leads to reduced proliferation and therefore provide a novel pharmacological target for CML and AML, the latter of which remains difficult to treat.

Age-Related Changes in Skeletal Muscle of Cattle.

Sarcopenia, the age-related loss of muscle mass and strength, is a multifactorial condition that represents a major healthcare concern for the elderly population. Although its morphologic features have been extensively studied in humans, animal models, and domestic and wild animals, only a few reports about spontaneous sarcopenia exist in other long-lived animals. In this work, muscle samples from 60 healthy Podolica-breed old cows (aged 15-23 years) were examined and compared with muscle samples from 10 young cows (3-6 years old). Frozen sections were studied through standard histologic and histoenzymatic procedures, as well as by immunohistochemistry, immunofluorescence, and Western blot analysis. The most prominent age-related myopathic features seen in the studied material included angular fiber atrophy (90% of cases), mitochondrial alterations (ragged red fibers, 70%; COX-negative fibers, 60%), presence of vacuolated fibers (75%), lymphocytic (predominantly CD8+) inflammation (40%), and type II selective fiber atrophy (40%). Immunohistochemistry revealed increased expression of major histocompatibility complex I in 36 cases (60%) and sarcoplasmic accumulations of ß-amyloid precursor protein-positive material in 18 cases (30%). In aged cows, muscle atrophy was associated with accumulation of myostatin. Western blot analysis indicated increased amount of both proteins-myostatin and ß-amyloid precursor protein-in muscles of aged animals compared with controls. These findings confirm the presence of age-related morphologic changes in cows similar to human sarcopenia and underline the possible role of amyloid deposition and subsequent inflammation in muscle senescence.

Reduced level of synapsin I protein in the rat striatum after intraventricular administration of proteasome inhibitors: preliminary studies.

BACKGROUND: We have recently described changes present in nigrostriatal terminals after intraperitoneal administration of MG-132 and changes that occur in the walls of the rat lateral ventricle after intraventricular administration of MG-132, lactacystin and epoxomicin - different classes of proteasome inhibitors. Substances that inhibit ubiquitin-proteasome system (UPS) activity, are intensively studied due to their potential role as novel therapeutic strategies in the treatment of cancer and ischaemia-reperfusion injury in the brain. The aim of this study is to determine the influence of intraventricular administration of MG-132, lactacystin and epoxomicin on the level in the rat striatum synapsin I - one of the most prominent neuron-specific phosphoproteins in the brain. MATERIALS AND METHODS AND RESULTS: Two weeks after administration of studied proteasome inhibitors, substantial reduction (up to 80%) of synapsin I was ob-served in the rat striatum. Because neurons, and especially dopaminergic ones, are sensitive to the depletion of proteasome function, we assume that observed synapsin I decrease may reflect changes in population of striatal neurons and/or nigrostriatal terminals. CONCLUSIONS: Understanding of cellular mechanisms standing behind our findings needs further studies, and could provide valuable contribution to the discussion on the mechanisms linking UPS inhibition and survival of neurons.

Myostatin and its precursor protein are increased in the skeletal muscle of patients with Type-II muscle fibre atrophy.

Preferential atrophy of Type-II muscle fibres occurs in several clinical situations, including cachexia, muscle disuse, chronic glucocorticoid treatment, remote neoplasia, and sometimes as an aspect of recent-denervation. For the patient, the Type-II atrophy itself might be unfavourable (as a glucocorticoid side-effect) or favourable (survivalistic via the muscle-alanine liver-gluconeogenesis pathway in starvation). The cellular mechanisms underlying Type-II fibre atrophy are unclear. Myostatin (Mstn) is physiologically a negative regulator of muscle mass and strength. In this study we evaluated a possible role of Mstn in Type-II fibre atrophy in human muscle. Mstn and Mstn precursor protein (MstnPP) were studied in 10-muscle biopsies containing Type-II fibre atrophy and in 17 disease and normal control muscle biopsies. When comparison was made with normal control fibres, we found the following: 1) by immunocytochemistry, diffusely increased Mstn/MstnPP in the atrophic Type-II muscle fibres; 2) by immunoblots, Mstn/MstnPP increased individually; 3) by RT-PCR, no increase in MstnPP mRNA. In conclusion, our results a) suggest that Mstn/ /MstnPP might play a role in the pathogenic cascade of Type-II muscle fibre atrophy; b) broaden our previously-described associations of Mstn in human muscle pathology, and c) could possibly lead to clinical prevention when Type-II muscle fibre atrophy is unfavourable, for instance in glucocorticoid therapy.

Distribution of neuronal nitric oxide synthase (nNOS)-immunoreactive elements in the rabbit piriform cortex.

The piriform cortex (PC), the primary olfactory cortex, is involved in the processes of learning and stress response and possibly plays an important role in epileptogenic activity. The results of several recent studies suggest that those PC neurons that contain neuronal nitric oxide synthase (nNOS) may play a key role during spatial learning and in the modulation of initiation, propagation and generalisation of seizures in various experimental models and may influence neuronal vulnerability after epileptic insults. The aim of this study was to characterise the pattern of distribution and morphology of nNOS-immunoreactive elements in PC of the adult rabbit. The co-localisation of nNOS and calretinin (CR) was also studied. The pattern of nNOS-ir within the rabbit PC is similar to that described previously in other mammals. The morphology of nNOS-ir elements, namely varicose fibres and Cajal-Retzius cells, suggest that NO has an important influence on PC function. Surprisingly, in the rabbit PC nNOS-ir elements show a very low level of co-localisation with CR-ir.

A case of multiple abnormalities of the azygos venous system: a praeaortic interazygos vein.

The posterior thoracic wall, an area drained by the azygos venous system, is a common site for surgical intervention. Since the venous part of the cardiovascular system is subject to most common variation, abnormalities in the azygos venous system are often reported. Some of the anatomical variants have significant clinical implications for computed tomography image assessment and mediastinal surgery. During dissection of the posterior mediastinum in a 76 year-old Caucasian male cadaver we found a rare variation in the azygos venous system. The hemiazygos vein drained the left 9th to 11th left posterior intercostal veins. While passing ventrally to the aorta at the level of the body of the eighth thoracic vertebra it was joined by two separate vessels found to be the continuations of the 7th and 8th left posterior intercostal veins. The resultant dilated vessel, termed the "interazygos vein", then opened into the azygos vein on the right side of the vertebral column. Variation in the azygos venous system has often been reported, but the abnormality observed by us appears to be extremely rare. The interazygos vein passing ventrally to the aorta may mimic enlarged lymph nodes and cause misinterpretation of a computed tomography image or, if accidentally damaged during mediastinal surgery, may lead to intraoperative haemorrhage. To the best of our knowledge this report provides new data of potential clinical significance.

Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1).

Three isoforms of a vesicular glutamate transporter (VGLUT1-3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down-regulation of VGLUT1 transporter on anxiety, depressive-like behaviour and learning. The behavioural phenotype of VGLUT1-heterozygous mice (C57BL/6) was compared to wild-type (WT) littermates. Moreover, VGLUT1-3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1-heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light-dark exploration test and depressive-like behaviour in the forced swimming test: no differences were shown in the elevated plus-maze model of anxiety. In the novel object recognition test, VGLUT1(+/-) mice showed normal short-term but impaired long-term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1-mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.

Parkin and its association with alpha-synuclein and AbetaPP in inclusion-body myositis and AbetaPP-overexpressing cultured human muscle fibers.

UNLABELLED: Parkin, an E3-ubiquitin ligase in the ubiquitin-proteasome system, facilitates degradation of alpha-synuclein and other proteins. Since ubiquitinated multiprotein-aggregates containing amyloid-beta (Abeta), alpha-synuclein, and other proteins, are characteristic of sporadic inclusion-body myositis (s-IBM) muscle fibers, we asked whether parkin might have a role in s-IBM pathogenesis. We studied the association of parkin with alpha-synuclein and Abeta-precursor protein (AbetaPP) in s-IBM muscle biopsies and in our IBM model based on overexpression of AbetaPP into cultured human muscle fibers. We report the following in s-IBM muscle fibers: a) parkin was increased 2.7 fold and accumulated in aggregates also containing Abeta and alpha-synuclein; b) alpha-synuclein was increased 6.3 fold; c) parkin physically associated with alpha-synuclein and AbetaPP; d) alpha-synuclein and AbetaPP were ubiquitinated. In the IBM model: a) parkin was increased 2.7 fold, b) it associated with alpha-synuclein and AbetaPP. CONCLUSION: 1. This is the first demonstration that in a human muscle disease alpha-synuclein associates with parkin, and might be ubiquitinated by it. 2. The small increase of parkin relative to the much larger increase of alpha-synuclein might be insufficient to secure complete ubiquitination and consequent degradation of alpha-syn. 3. AbetaPP might be a novel substrate of parkin.

Quantitative analysis of influence of sevoflurane on the reactivity of microglial cells in the course of the experimental model of intracerebral haemorrhage.

BACKGROUND: Microglial cells play an important role in the pathophysiology of intracerebral haemorrhage. We have examined the possible influence of sevoflurane on the reactivity of microglial cells during intracranial haemorrhage. METHODS: Forty adult male rats were divided into two groups. All animals were anaesthetized with fentanyl, dehydrobenzperidol and midazolam. In the experimental group animals additionally received sevoflurane 2.2 vol% end-tidal concentration. Intracranial haemorrhage was produced through infusion of blood into the striatum. The microglial cell population (numerical density of immunoreactive cells and their distribution) was assessed on days 1, 3, 7, 14 and 21 after producing a haematoma using antibodies OX42 and OX6. RESULTS: In the control group significant differences in the density of OX42-ir cells between 3rd and 7th (81.86 vs. 129.99) (95% CI: -77.99 to -18.25, P = 0.0035) and between 14th and 21st (105.36 vs. 63.81) (95% CI: 13.21 to 69.89, P = 0.006) survival days were observed. However, significant increase of percentage of amoeboid OX42-ir cells between 3rd and 7th (0.98 vs. 48.71) (95% CI: -52.17 to -43.30, P = 0.0001) and between 7th and 14th (48.71 vs. 58.47) (95% CI: -13.96 to -5.55, P = 0.0002) and then their decrease - between 14th and 21st (58.47 vs. 31.74) (95% CI: 22.52 to 30.93, P = 0.0001) days of observation were noted. In the sevoflurane groups OX42-ir cells were not found. On the 3rd day the density of OX6-ir cells in the sevoflurane group was significantly lower than that in the control group (12.39 vs. 34.57) (95% CI: -49.78 to -2.96, P = 0.02). The percentage of an amoeboid form of OX6-ir cells was significantly lower in the sevoflurane group than that in the control group (27.31 vs. 82.03) (95% CI: -72.52 to -36.92, P = 0.0001) (58.76 vs. 82.37) (95% CI: -38.81 to -8.41, P = 0.003) (42.87 vs. 81.55) (95% CI: -53.23 to -24.10, P = 0.0001) respectively for 3rd, 7th and 14th days of survival. CONCLUSION: Administration of sevoflurane during anaesthesia in animals with intracerebral haemorrhage evoked a decrease of activation of the microglial cells.

Implementation of X-ray fluorescence microscopy for investigation of elemental abnormalities in amyotrophic lateral sclerosis.

The abnormalities of metallochemical reactions may contribute to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). In the present work, an investigation of the elemental composition of the gray matter, nerve cells and white matter from spinal cord tissues representing three ALS cases and five non-ALS controls was performed. This was done with the use of the synchrotron microbeam X-ray fluorescence technique (micro-SRXRF). The following elements were detected in the tissue sections: P, S, Cl, K, Ca, Fe, Cu, Zn and Br. A higher accumulation of Cl, K, Ca, Zn and Br was observed in the nerve cell bodies than in the surrounding tissue. Contrary to all other elements, Zn accumulation was lower in the white matter areas than in the gray matter ones. The results of quantitative analysis showed that there were no general abnormalities in the elemental accumulation between the ALS and the control group. However, for individual ALS cases such abnormalities were observed for the nerve cells. We also demonstrated differences in the elemental accumulation between the analyzed ALS cases.

Increased expression of Noga-A in ALS muscle biopsies is not unique for this disease.

Nogo (RTN4) belongs to the reticulon (RTN) family of integral membrane proteins. RTN4A (Nogo-A), RTN4B (Nogo-B) and RTN4C (Nogo-C) are isoforms of RTN4. In the gastrocnemius muscle of transgenic mice bearing an SOD1 mutation ("ALS model"), increased Nogo-A mRNA and protein was reported, and similar changes were reported in muscle biopsies of patients with amyotrophic lateral sclerosis (ALS) but not with peripheral neuropathy or primary muscle diseases, leading to the proposal that Nogo-A in skeletal muscle is a new specific molecular marker of ALS. Here we report, based on studies of muscle biopsies from patients with ALS, peripheral neuropathies, polymyositis, dermatomyositis and morphologically nonspecific myopathies that, in addition of strong Nogo-A immunoreactivity within apparently-denervated small angular fibers in ALS and peripheral neuropathies, Nogo-A was strongly immunoreactive within desmin-positive regenerating muscle fibers in various myopathies, and its expression on immunoblots was increased in all those neuromuscular diseases. In conclusion, we have found that the presence of Nogo-A in diseased human muscle biopsies is not limited to ALS.

The influence of sevoflurane on the reactivity of astrocytes in the course of the experimental intracerebral haemorrhage in rat.

40 adult Wistar rats were divided into two groups depending on the applied anaesthesia. In both groups animals were generally anaesthetized with fentanyl, dehydrobenzperidol administered intraperitoneally and midazolam given intramuscularly. In the second group (SEVO) animals received sevoflurane of 2.2 vol% end-tidal concentration. Intracerebral haematoma was produced through infusion of 100 microl of autologous blood into the striatum. Each group was divided into five subgroups depending on the length of survival period: 1, 3, 7, 14, 21 days. The astrocytic population was studied by means of anti-GFAP staining. Stereological analysis was applied to estimate the numerical density of immunoreactive cells and the distribution of their types. On 7th day of observation the density of GFAP-immunoreactive astrocytes in SEVO was lower (p<0,05) than that in the control group. In the control group, the increase (p<0.05) of per cent of activated astrocytes between the 1st and 3rd survival day was noted, which remained at this level till the end of observation. In SEVO group, the increase (p<0.05) of per cent of activated astrocytes between the 3rd and 7th day and the decrease (p<0.05) between the 14th and 21st survival day were observed. During days of observation the per cent of activated astrocytes was lower (p<0.05) in the SEVO group than that in the control group. Administration of sevoflurane during anaesthesia to animals with intracerebral haemorrhage has evoked not only the delay of the activation of astrocytes but also decrease in its level.

Open field stress and neurons containing calcium-binding proteins in the piriform cortex of the rat.

In the present study we wanted to check whether the expression of the c-Fos protein (the marker of cellular activity) appears in cells containing calcium-binding proteins (CaBPs) in animals exposed to the open field test. Eight adult Wistar rats were examined. In the first step the open field test was applied throughout 10 minutes. After perfusional fixation brains were frozen and cut on the cryostat in the coronal plane and stained with the standard immunohistochemical method. Sections were double stained for c-Fos and CaBPs: parvalbumin (PV), calbindin (CB), calretinin (CR). c-Fos positive cells were localized predominantly in layers II and III of the piriform cortex (PC). The double labeling study showed that neurons containing CaBPs are rarely c-Fos-immunoreactive. Often PV-positive and CB-positive fibers surround c-Fos-positive neurons in layers II and III in a form of a basket. It seems that cells containing CaBPs are not directly involved in the response to aversive stimuli but cells containing those calcium-binding proteins might influence directly c-Fos positive neurons of PC.

Apoptosis in the course of experimetal intracerebral haemorrhage in the rat.

Intracerebral haematoma was produced in 25 adult rats by infusion of 100 microl of autologous blood into the striatum. The animals' brains were removed at 1, 3, 7, 14 and 21 days after production of the haematoma. The TUNEL method was used to detect DNA fragmentation and TUNEL-positive cells were qualified. TUNEL-positive cells were already found on the first day of observation and were present for three weeks after haematoma production. These results provide evidence that programmed cell death is associated with intracerebral haemorrhage.