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.

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.

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.

Mutations in the rod domain of keratin 2e in patients with ichthyosis bullosa of Siemens.

Ichthyosis bullosa of Siemens (IBS) is an autosomal dominant skin disorder that resembles epidermolytic hyperkeratosis (EHK). We have identified mutations in two families originally diagnosed with EHK and in four families diagnosed with IBS at the same codon in the highly conserved carboxy terminal of the rod domain of keratin 2e, thus revealing a mutational hot spot. Our results allow a differential diagnosis to be made between IBS and EHK at the genetic level and we suggest that patients diagnosed with EHK, but lacking keratin K1 or K10 mutations, should be re-examined for mutations in their K2e genes.

Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b.

The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b-/- mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, approximately 25% of MK6a/b-/- mice survive to adulthood. Remarkably, the surviving MK6a/b-/- mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b-/- mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.

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.

Delayed wound healing in keratin 6a knockout mice.

Keratin 6 (K6) expression in the epidermis has two components: constitutive expression in the innermost layer of the outer root sheath (ORS) of hair follicles and inducible expression in the interfollicular epidermis in response to stressful stimuli such as wounding. Mice express two K6 isoforms, MK6a and MK6b. To gain insight into the functional significance of these isoforms, we generated MK6a-deficient mice through mouse embryonic stem cell technology. Upon wounding, MK6a was induced in the outer ORS and the interfollicular epidermis including the basal cell layer of MK6a(+/+) mice, whereas MK6b induction in MK6a(-/-) mice was restricted to the suprabasal layers of the epidermis. After superficial wounding of the epidermis by tape stripping, MK6a(-/-) mice showed a delay in reepithelialization from the hair follicle. However, the healing of full-thickness skin wounds was not impaired in MK6a(-/-) animals. Migration and proliferation of MK6a(-/-) keratinocytes were not impaired in vitro. Furthermore, the migrating and the proliferating keratinocytes of full-thickness wounds in MK6a(-/-) animals expressed neither MK6a nor MK6b. These data indicate that MK6a does not play a major role in keratinocyte proliferation or migration but point to a role in the activation of follicular keratinocytes after wounding. This study represents the first report of a keratin null mutation that results in a wound healing defect.

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.

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.

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.

Arachidonic acid, a growth signal in murine P815 mastocytoma cells.

Evidence is presented that inducing P815 murine mastocytoma cells to grow with serum activates a Ca(2+)-stimulated phospholipase A2 and the rapid release of arachidonic acid by the cells. Slower growth was also maintained by arachidonic acid or its immediate precursors or by diacylglycerols when bovine serum albumin replaced the serum. Together, arachidonic acid and 1-oleoyl-2-acetylglycerol stimulated growth at the same rate as 10% serum consistent with a role for both arachidonic acid and protein kinase C in the response to serum. Arresting cell growth with N6,O2'-dibutyryladenosine 3',5'-cyclic phosphate and theophylline inhibited the release of arachidonic acid in response to serum, suggesting that cyclic AMP prevents phospholipase activation as one of its pleiotypic effects on growth. Attempts to demonstrate metabolism of [3H]arachidonic acid to eicosanoids in serum-treated P815 cells by high-performance liquid chromatography or thin layer chromatography were unsuccessful, with the major products being phospholipids and triacylglycerol. Incubating digitonin-permeabilized P815 cells with [gamma-32P]ATP and arachidonic acid rapidly increased the phosphorylation of some proteins in the cells, especially the M(r) 135,000 and M(r) 44,000 proteins which were considerably more phosphorylated than the rest. Phosphorylation of these proteins was not prevented by several inhibitors of protein kinase C, nor was it increased by diacylglycerols or phorbol ester, suggesting that arachidonic acid activates a growth-related protein kinase other than protein kinase C in P815 cells. The possibility that some polyunsaturated fatty acids may promote tumor cell growth by stimulating protein phosphorylation is considered.

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.

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.

Inhibition of turnip yellow mosaic virus synthesis by pyrimidine analogues.

The pyrimidine analogues 2-thiouracil, 2-thiouridine, 6-azauracil and 6-azauridine all inhibited the synthesis of turnip yellow mosaic virus (TYMV) and increased the synthesis of empty virus protein shells in infected Chinese cabbage leaf discs. Uracil and uridine reversed these effects. 2-Thiouracil also reduced the UTP pool in TYMV infected leaf discs. The results are consistent with the suggestion that these analogues or their in vivo derivatives affect virus synthesis by inhibiting the biosynthesis of uridylic acid, possibly by inhibiting orotidylic acid decarboxylase.

Effects of kinetin on phosphorylation of leaf membrane proteins.

Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.

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.