The Potassium Channel Kv1.5 Expression Alters During Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic, inflammatory, neurodegenerative disease with an autoimmune component. It was suggested that potassium channels, which are involved in crucial biological functions may have a role in different diseases, including MS and its animal model, experimental autoimmune encephalomyelitis (EAE). It was shown that voltage-gated potassium channels Kv1.5 are responsible for fine-tuning in the immune physiology and influence proliferation and differentiation in microglia and astrocytes. Here, we explored the cellular distribution of the Kv1.5 channel, together with its transcript and protein expression in the male rat spinal cord during different stages of EAE. Our results reveal a decrease of Kv1.5 transcript and protein level at the peak of disease, where massive infiltration of myeloid cells occurs, together with reactive astrogliosis and demyelination. Also, we revealed that the presence of this channel is not found in infiltrating macrophages/microglia during EAE. It is interesting to note that Kv1.5 channel is expressed only in resting microglia in the naïve animals. Predominant expression of Kv1.5 channel was found in the astrocytes in all experimental groups, while some vimentin+ cells, resembling macrophages, are devoid of Kv1.5 expression. Our results point to the possible link between Kv1.5 channel and the pathophysiological processes in EAE.

17ß-Estradiol upregulates ecto-5'-nucleotidase (CD73) in hippocampal synaptosomes of female rats through action mediated by estrogen receptor-α and -ß.

17ß-Estradiol (E2) crucially affects several processes in the hippocampus of both sexes. E2 acts upon estradiol receptors ERα and ERß, influencing target gene expression and/or modulates intracellular signaling cascades. Another potent modulator of hippocampal function is nucleoside adenosine, the final product of ectonucleotidase cascade, enzymes which hydrolyze extracellular ATP to adenosine. The last and rate-limiting step of the hydrolysis is catalyzed by membrane-bound ecto-5'-nucleotidase (eN). Previous findings obtained on adenosine metabolism in brain suggest that eN may be modulated by ovarian steroids. Therefore, the present study reports that the activity and protein abundance of membrane-bound eN fluctuates across the estrus cycle in the hippocampal synaptosomes of female rats. Further, we analyzed the role of E2 and its intracellular receptors on the expression of eN in ovariectomized females. We found that E2 upregulated eN activity and protein abundance in the hippocampal synaptosomes. Application of nonspecific ER antagonist, ICI 182,780 and selective ERα and ERß agonists, PPT and DPN, respectively, demonstrated the involvement of both receptor subtypes in observed actions. Selective ERα receptor agonist, PPT, induced upregulation of both the protein level and activity of eN, while application of selective ERß receptor agonist, DPN, increased only the activity of eN. In both cases, E2 entered into the intracellular compartment and activated ER(s), which was demonstrated by membrane impermeable E2-BSA conjugate. Together these results imply that E2-induced effects on connectivity and functional properties of the hippocampal synapses may be in part mediated through observed effect on eN.

Ki-67 expression in oral lichen planus.

PURPOSE: The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. This is of particular interest for the analysis of the proliferation rates of malignant tumors. The aim of this study was to investigate the malignant potential of oral lichen planus (OLP) on the basis of expression of Ki-67 in healthy individuals (HI), patients with OLP and patients with squamous cell carcinoma (SCC), and to see for any potential interdependence between Ki-67 expression and different clinical and histopathological parameters in OLP. METHODS: Immunohistochemistry for Ki-67 was carried out using an avidin-biotin peroxidase complex method. RESULTS: Ki-67 was more expressed in keratinocytes and lymphocytes of OLP patients compared with HI, but less compared with patients with SCC. Keratinocytes and lymphocytes stained with Ki-67 in OLP patients were significantly higher in males, and in OLP specimens showed less developed civatte bodies (CB) and thickening of the basal membrane (TBM). CONCLUSION: Ki-67 may not serve as prognostic biomarker in oral cancer development from the initially diagnosed OLP, but it could help selecting patients with higher need of follow up for prevention of malignancy.

The cortical stab injury induces beading of fibers expressing ecto-nucleoside triphosphate diphosphohydrolase 3.

The ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), an enzyme involved in degradation of extracellular adenosine triphosphate (ATP), is expressed on nerve fibers in different brain regions, including cortex. Here we studied the expression and role of this enzyme after unilateral cortical stab injury in rats. In cortical sections of control rats, NTPDase3 immunoreactivity was associated with two types of fibers: thin processes, occasionally with small mushroom-like protrusions and slightly thicker fibers with more pronounced and more frequent varicosities, whereas immunopositive neuronal perycaria were never observed. Although NTPDase3-positive thin processes and thicker fibers, by general appearance, size and shape, could be dendrites and axons, respectively, they were never immunopositive for microtubule associated protein-2 or neurofilament H subunit. Cortical stab injury induced rapid (within 4 hours) focal varicose swelling that evolved over time to prominent beading of NTPDase3-positive fibers. The NTPDase3-positive fibers in all experimental groups also abundantly express NTPDase1, ecto-5'-nucleotidase and P2X2 receptor channels. Because the brain injury causes a massive ATP release, it is reasonable to conclude that purinoreceptors and ectonucleotidases play an important role in the process of neuritic beading.

Pattern of glial fibrillary acidic protein expression following kainate-induced cerebellar lesion in rats.

In the present study glial fibrillary acidic protein (GFAP) expression was assessed following intravermian injection of kainic acid (KA) or physiological saline to adult rat cerebellum. After 2- to 30-day recovery period, free-floating sections cut with a microtome were obtained and were proccessed for immunocytochemistry against GFAP. Injection of both kainate and physiological saline elicited significant astrogliotic reaction, i.e. in the area around the lesion thick GFAP-positive Bergmann fibers with typical orientation appeared in the molecular and hypertrophied astrocytes abundantly appeared in the granular layer. However, following kainate intoxication lesion was not surrounded by typical demarcation glial scar during 30-day recovery period in contrast to the appearance of usual glial scar in the group injected with physiological saline, as early as 7-day postlesion. Preserved spatial organization of Bergmann fibers and the absence of typical demarcating glial scar after kainate-induced cerebellar lesion suggest distinct pattern of astrogliosis that presents an interesting model system to study the importance of glial scar in the recovery after ischemic brain insults.

Developmental profile of NTPDase activity in synaptic plasma membranes isolated from rat cerebral cortex.

In the present study the developmental profile of ATP-hydrolyzing activity promoted by NTPDase 1, its kinetic properties and the enzyme protein abundance associated with synaptic plasma membrane from rat cerebral cortex were characterized. NTPDase 1 activity increased from birth to day 30; afterwards it decreased and remained unchanged from adulthood (90 days) to senescence (365 days). Kinetic analysis revealed that enzyme exhibited the highest specific activity at day 30 and highest apparent affinity for ATP at day 365; however, V(max)/K(m) values remained unchanged for each age studied. Immunoblot analysis demonstrated that relative abundance of NTPDase 1 is highest at day 15 during ontogeny. The discrepancy between maximum enzyme activity and maximum enzyme protein abundance indicates that NTPDase 1 may have an additional role during development.

Spatio-temporal changes in neurofilament proteins immunoreactivity following kainate-induced cerebellar lesion in rats.

1. Spatio-temporal changes in phosphorylated (pNFP) and nonphosphorylated (npNFP) neurofilament proteins were assessed immunocytochemicaly in adult rat cerebellum, 2-30 days following unilateral injection of kainic acid (KA) or physiological saline (s.c.). 2. Analysis of the staining intensity and pattern demonstrated that injection of both KA and physiological saline elicited significant and long-lasting increase of pNFP and npNFP immunoreactivity, at the ipsilateral, and to lesser extent at the contralateral side of lesion. 3. Kainate intoxication induced abundant expression of pNFP and npNFP in cerebellar white matter, as well as in all layers of perilesioned cortex. Higher pNFP expression was evidenced in the Purkinje cell layer, particularly at cell bodies, initial segments, and proximal dendrites, which normally do not contain pNFP. In addition, synaptophysin immunocytochemistry was used as a marker of synaptogenesis and plasticity. 4. Spatio-temporal pattern of NFP and synaptophysin expression suggests that perilesioned cortex undergoes dynamic changes following brain demage and possess a reparative capacity to abridge the consequences of brain trauma.

Ecto-ATPase and ecto-ATP-diphosphohydrolase are co-localized in rat hippocampal and caudate nucleus synaptic plasma membranes.

Enzymes that hydrolyze extracellular ATP, i.e. ecto-ATPase and ecto-ATP diphosphohydrolase (ATPDase), can be differentiated by ability of the latter to hydrolyze ADP and by slightly different kinetic properties of the two enzymes. Synaptic plasma membrane fractions isolated from rat hippocampus and caudate nucleus exhibit ADP-hydrolyzing activity, as revealed by the enzyme assay, and the presence of ecto-ATPase protein, as revealed by immunological identification on Western blot. These findings indicate that both enzymes are co-expressed in the synaptic membrane compartment of hippocampal and caudate nucleus neurons. Kinetic analysis was performed to determine the relative contribution of each enzyme to the total ATP-hydrolyzing activity, while an inhibition study was carried out in order to exclude the interference of other nonspecific ATPase and phosphatase activities. Based on the kinetic properties, sensitivity to inhibitors and V(ATP)/V(ADP) ratio of about 2, we concluded that a substantial portion of ATP-hydrolyzing activity in both synaptic membrane preparations can be ascribed to the catalytic action of ATPDase. On the other hand, the highest catalytic efficacy when ATP is the substrate and the greater abundance of ecto-ATPase protein in caudate nucleus preparation suggest that the relative contribution of ecto-ATPase to the total ATP-hydrolyzing activity in the caudate nucleus is higher than in the hippocampus.

Ontogenetic profile of ecto-ATPase activity in rat hippocampal and caudate nucleus synaptic plasma membrane fractions.

An ontogenetic study of ecto-ATPase activity and the content of enzyme proteins was assessed in the caudate nucleus and hippocampal synaptic plasma membranes isolated from rats at various ages (15, 30, 90, 180 and 365 days). The ontogenetic profile revealed that the enzyme activities in both brain areas were the highest on day 30 and 365, while the ecto-ATPase protein abundance was the highest on day 15 after birth. Possible explanation for obtained ontogenetic profile and the discrepancy between activity and abundance may reside in the fact that ecto-ATPase during development could exert additional roles other than those related to metabolism of ATP. It is likely that ecto-ATPase, regulating the concentration of ATP and adenosine in synaptic cleft, has important role in the processes of brain development and aging.

Effect of steroid hormone deprivation on the expression of ecto-ATPase in distinct brain regions of female rats.

Abundant evidence indicates that ATP and adenosine act as neurotransmitters or co-transmitters, influencing nerve cell physiology in various ways. Therefore, regulation of ATP-metabolizing enzymes is essential for the normal development and function of neuronal tissue. In the present study we have examined the effect of gonadal (OVX) or adrenal (ADX) steroid hormone deprivation on the activity and expression of synaptic membrane ecto-ATPase in three extrahypothalamic brain areas of female rats, primarily not associated with reproductive function. It was shown that OVX significantly increased ecto-ATPase activity and the relative abundance of this enzyme in the hippocampal (Hip) and caudate nucleus (CN), but not in brain stem (BS) membrane preparations. ADX was followed by an upregulation of the enzyme activity and its relative abundance in all the brain areas investigated. The highest enzyme activity and the most profound effects of OVX and ADX were detected in the CN. The results obtained indicate that ADX and OVX upregulate the expression of ecto-ATPase, potentiating the production of adenosine in synaptic cleft thus modulating the activity of numerous neurotransmitter systems in distinct areas of the CNS.

Estradiol affect Na-dependent Ca2+ efflux from synaptosomal mitochondria.

The effects of gonadal steroid hormone, 17beta-estradiol (E2), in vitro on rat brain mitochondria Ca2+ movement were investigated. Intrasynaptosomal mitochondria Ca2+ uptake via an energy-driven Ca2+ uniporter have Km = 112.73 +/- 7.3 micromol x l(-1) and Vmax = 21.97 +/- 1.7 nmol 45Ca2+ mg(-1). Ca2+ release trough a Na+/Ca2+ antiporter was measured with a Km for Na+ of 43.7 +/- 2.6 mmol x l(-1), and Vmax of 1.5 +/- 0.3 nmol 45Ca2+ mg(-1). Addition of estradiol in preincubation mixture did not affect the uptake of Ca2+ mediated by the ruthenium red-sensitive uniporter, while it produced biphasic effect on Na-dependent Ca2+ efflux. Estradiol at concentrations up to 1 nmol x l(-1) decreased the efflux significantly (63% inhibition with respect to the control), and at concentrations above 10 nmol x l(-1) increased it exponentially. The maximum inhibiting concentration of estradiol (0.5 nmol x l(-1)) increased the affinity of the uniporter (Km reduced by about 30%), without affecting significantly the capacity (Vmax) for Na+. The results presented suggest that estradiol inhibits Na-dependent Ca2+ efflux from mitochondria and acts on mitochondrial retention of Ca2+, which may modulate mitochondrial and consequently synaptosomal content of Ca2+, and in this way exerts its role in the homeostasis of calcium in nerve terminals.

Prevention and recovery of CuSO4-induced inhibition of Na+/K+ -ATPase and Mg2+ -ATPase in rat brain synaptosomes by EDTA.

Enzymatic activities of Na+/K+-ATPase and Mg2+ -ATPase from rat brain synaptic plasma membrane were studied in the absence and presence of EDTA. The aim of the study was to examine the ability of this strong chelator to prevent and recover the CuSO4-induced inhibition. The influence of experimentally added CuSO4 and EDTA on MgATP2- complex and 'free' Cu2+ concentrations in the reaction mixture was calculated and discussed. CuSO4 induced dose-dependent inhibition of both enzymes in the absence and presence of 1 mM EDTA. In the absence of EDTA, the IC50 values of Cu2+, as calculated from the experimental curves, were 5.9x10(-7) M for Na+/K+ -ATPase and 3.6x10(-6) M for Mg2+ -ATPase. One millimolar EDTA prevented the enzyme inhibition induced by CuSO4, but also reversed the inhibited activity, in a concentration-dependent manner, following exposure of the enzymes to the metal ion, by lowering 'free' Cu2+ concentration. Kinetic analysis showed that CuSO4 inhibits both the Na+/K+ -ATPase and Mg2+ -ATPase, by reducing their maximum enzymatic velocities (Vmax), rather than apparent affinity for substrate MgATP2- (K0.5), implying the noncompetitive nature of enzyme inhibition induced by the metal. The kinetic analysis also confirmed two distinct Mg2+ -ATPase subtypes activated in the presence of low and high MgATP2- concentrations. K0.5 and Vmax were calculated using a computer-based program. The results of calculation showed that MgATP2- concentration in the kinetic experiments exceeded three times the apparent K0.5 value for the enzyme activation.

Properties of Mg(2+)-ATPase rat brain synaptic plasma membranes.

In the present study distribution and enzymatic properties of ecto-Mg(2+)-ATPase were determined in synaptic plasma membrane (SPM) preparations isolated from the hippocampus, caudate nucleus and whole brains of female rats. Western blot analysis using anti-ecto-Mg(2+)-ATPase antibody revealed the association of Mg(2+)-ATPase with SPM prepared from all the three brain sources, yet the enzyme was most abundant in caudate nucleus membranes, being 30% and 22% more abundant than in the hippocampal and whole brain tissue SPM, respectively. The evidence is also presented that kinetic properties of the brain Mg(2+)-ATPase are not under the control of circulating sex steroids. It was confirmed that the enzyme is activated by millimolar concentrations of Mg2+ and that it cannot be effectively inhibited by known ATPase inhibitors. The most pronounced differences in kinetic properties observed were 2.5 fold higher apparent affinity for ATP and 59% higher specific activity of Mg(2+)-ATPase of the caudate nucleus as compared with the enzyme from the hippocampus. On the other hand, the apparent enzyme affinity for Mg2+ was almost equal in all SPM preparations tested. Taken together, our results show that ecto-Mg(2+)-ATPase is not uniformly distributed and differs in respect to affinity for ATP in rat brain regions, thus indicating its substantial role in the process of signal transduction via controlling the levels of extracellular ATP.

Influence of pyridine and urea on the rat brain ATPase activity.

The neurotoxicity of pyridine and urea was investigated in respect to their ability to alter the activity of synaptosomal membrane Na+/K(+)-ATPase and Mg(2+)-ATPase. In vitro treatment with pyridine and urea stimulated Na+/K(+)-ATPase activity in a dose-dependent manner up to 40% and 60%, respectively. Mg(2+)-ATPase activity increased up to 40% after pyridine treatment, while urea had no effect at all. The neuroactive potencies of pyridine and urea were evaluated by estimating parameters Km and delta Vmax for enzyme stimulation, as well as Hill coefficient to estimate the levels of cooperativity for pyridine and urea binding. The results suggest that pyridine stimulates both enzymes, probably by interacting with some neuronal membrane components, and altering the lipid micro-environment of the ATPases. In contrast, urea stimulates the Na+/K(+)-ATPase only, assumingly by acting on it directly or via some other regulatory mechanism. Stimulation of Na+/K(+)-ATPase and Mg(2+)-ATPase by the substances tested and subsequent alteration of neuronal cell functioning could contribute to the CNS dysfunction upon chronic exposure to pyridine and urea.

A simple morphological score for the quality control of platelet concentrates.

The percentage of platelets that retain discoid form correlate with their post-transfusion viability and therefore they may act as a simple indicator for the quality of the stored platelet concentrates (PCs). The morphological score (MS) of platelets, given by Kunicki, requires the index of different forms of platelets: discoid, spherical, starlike and bizarre. In the present study, we intended to clarify whether the morphological score of discoid platelets alone could serve as a method for the estimation of PCs quality. During the first five days after preparation, samples of standard platelet concentrates (SPCs) and of leucodepleted platelet concentrates (LDPCs) were analyzed morphologically by immersion light microscopy using smears stained by the May-Grünwald-Giemsa method. In addition to the MS, an original Modified Morphological Score (MMS), for a population of 200 platelets, was used to count platelets with the discoid shape. The mean MMS ranged (first to fifth day) from 192.6 to 166.6 with SPCs and from 197.2 to 185.4 with LDPCs. There is a significant difference (ANOVA) between: (a) PCs prepared by different techniques (p < 0.01); (b) the quality of the same PCs during the storage (p < 0.0001).

Estradiol in vitro modulates Na+-dependent Ca2+ uptake by synaptic plasma membrane vesicles of rat brain regions.

Membrane vesicles loaded with [Na+], prepared from synaptosomal plasma membranes (SPM) of whole brains (WB), hippocampi (Hip) and caudate nuclei (NC) of female rats, were used to study Na+ -dependent Ca2+ transport across SPM vesicles under the influence of 17beta-estradiol (E2) in vitro. In concentrations near to physiologic, E2 significantly increased 45Ca2+ uptake by SPM vesicles from all the brain tissues investigated. The maximum increase was observed for WB (21%) and Hip (33%) at 10(-9) mol/l, and for NC (31%) at 5 x 10(-9) mol/l of E2. These results (a) confirm our earlier finding that E2 in vitro modulates Na+-dependent Ca2+ transport across synaptosomal membrane in rat brain regions, and (b) suggest Na+/Ca2+ exchange as principal mechanism of the E2-stimulated Na-dependent Ca2+ uptake by membrane vesicles. The involvement of any ATPases as possible mediators is discussed.

Biochemical characterization of the hippocampal and striatal Na,K-ATPase reveals striking differences in kinetic properties.

The activities and basic enzymatic properties of Na,K-ATPase were examined in synaptosomal plasma membranes (SPM) prepared from rat hippocampus and striatum. A kinetic analysis showed profound differences in apparent affinities for ATP (Km) between hippocampal (1.21 mmol/l) and striatal (0.76 mmol/l) enzyme preparations, as well as in the corresponding Vmax values. However, physiological efficiencies were almost the same. The complex pattern of dose-response curves to ouabain indicated the presence of two high-affinity forms of Na,K-ATPase in the striatum ("very high-": Ki = 3.73 x 10(-8) mol/l and "high-": Ki = 4.21 x 10(-5) mol/l), and one high affinity form in the hippocampus (Ki = 6.6 x 10(-7) mol/l). In addition, both SPM preparations contained one low affinity form with similar Ki. The "very high-affinity" form had positive cooperativity for ouabain inhibition of Na,K-ATPase activity, in contrast to "high" and "low-affinity" forms, which exhibited negative cooperativity. The respective contributions of ouabain-sensitive forms to the total activity were estimated as 22%, 46%, 19% for the striatum and 36%, 45% for the hippocampus. These data clearly demonstrate striking differences in kinetic properties of the hippocampal and striatal Na,K-ATPase that may be due to the isoenzyme diversity and adaptation to specific physiological demands of the examined rat brain regions.

17beta-estradiol in vitro affects Na-dependent and depolarization-induced Ca2+ transport in rat brain synaptosomes.

Effects of 17beta-estradiol (E2) in vitro on Na-dependent Ca2+ efflux from, and depolarization-induced Ca2+ uptake into, the nerve cell were studied with the use of synaptosomes isolated from the brain stem, mesencephalic reticular formation (MRF), caudate nucleus and the hippocampus of long-term ovariectomized adult female rats. It was found that E2 (1) at a concentration of 10 nM or lower, stimulates Na-dependent Ca2+ efflux in the caudate nucleus and hippocampus, and does not affect the efflux in MRF and brain stem; (2) at concentrations above 10 nM has no effect on the Ca2+ efflux in any of the four structures investigated; and (3) produces a biphasic effect on the depolarization-induced Ca2+ uptake, increasing it in all structures except MRF at 10 nM concentration, and decreasing it at concentrations higher than 10 nM, irrespective of the structure investigated. These results suggest that E2, acting at extranuclear sites, modulates synaptic transmission via alterations of Ca2+ transport mechanisms in nerve endings.

[Cytomegalovirus transmission by fresh blood]. / Prenosenje citomegalovirusa svezom krvi.

Investigations included blood donors for extracorporeal circulation, as well as patients (12) before and after cardiopulmonary by-pass. CMV infection is a frequent consequence of multiple fresh blood transfusions in persons with aortocoronary "by-pass" in 25% operated patients. If a patient receives more than 10 transfusions of fresh blood, frequency of serologically positive reactions to CMV is found in approximately 40-60% operated patients. Investigations included 168 healthy individuals, voluntary blood donors, 18-30 years old, both male and female, from various backgrounds and with various occupations. It was found out that amount of antibodies against CMV for men was 42,1% in spring and 66,7% in winter, while the amount of antibodies against CMV for women was 70% in spring and 81% in winter. Complement fixation reaction method was applied.